Full-length serine protein kinase in brain and pancreas

ABSTRACT

The present invention relates to all facets of novel polynucleotides, the polypeptides they encode, antibodies and specific binding partners thereto, and their applications to research, diagnosis, drug discovery, therapy, clinical medicine, forensic science, pathology, and medicine, etc. The polynucleotides are expressed in brain and pancreas and are therefore useful in variety of ways, including, but not limited to, as molecular markers, as drug targets, and for detecting, diagnosing, staging, monitoring, prognosticating, preventing or treating, determining predisposition to, etc., diseases and conditions, especially relating to brain and pancreas.

[0001] This application is a continuation of U.S. application Ser. No.09/930,181, filed Aug. 16, 2001, which is hereby incorporated byreference in its entirety.

DESCRIPTION OF THE DRAWINGS

[0002] SEQ ID NOS. 1 and 2 show the nucleotide and amino acid sequencesof KSE336-1. SEQ ID NOS. 3 and 4 show the nucleotide and amino acidsequences of KSE336-2. The N-terminal amino acid sequence of KSE336-1 isshown in SEQ ID NOS 5 and 6. Promoter sequences for KSE336 are listed inSEQ ID NOS 7-14. SEQ ID NOS. 15 and 16 show substrate for serine kinaseactivity. The amino acid sequence of AJ006701 is shown in SEQ ID NO 17and the amino acid sequence of AF020089 is shown in SEQ ID NO 18. Theseare human cDNAs.

[0003]FIG. 1 shows the expression pattern of KSE 336. 1, adrenal gland;2, bone marrow; 3, brain; 4, colon; 5, heart; 6, intestine; 7, kidney;8, liver; 9, lung; 10, lymph node; 11, lymphocytes; 12, mammary gland;13, muscle; 14, ovary; 15, pancreas; 16, pituitary; 17, prostate; 18,skin; 19, spleen; 20, stomach; 21, testis; 22, thymus; 23, thyroid; 24,uterus.

[0004]FIG. 2 shows sequence comparisons between KSE336-1 (SEQ ID NO 2),KSE336-2 (SEQ ID NO 4), AJ00601 (SEQ ID NO 17), and AF020089 (SEQ ID NO18).

DESCRIPTION OF THE INVENTION

[0005] The present invention relates to all facets of novelpolynucleotides, the polypeptides they encode, antibodies and specificbinding partners thereto, and their applications to research, diagnosis,drug discovery, therapy, clinical medicine, forensic science andmedicine, etc. The polynucleotides are expressed in brain and pancreasand are therefore useful in variety of ways, including, but not limitedto, as molecular markers, as drug targets, and for detecting,diagnosing, staging, monitoring, prognosticating, preventing ortreating, determining predisposition to, etc., diseases and conditions,especially relating to brain and pancreas. The identification ofspecific genes, and groups of genes, expressed in pathwaysphysiologically relevant to brain and pancreas permits the definition offunctional and disease pathways, and the delineation of targets in thesepathways which are useful in diagnostic, therapeutic, and clinicalapplications. The present invention also relates to methods of using thepolynucleotides and related products (proteins, antibodies, etc.) inbusiness and computer-related methods, e.g., advertising, displaying,offering, selling, etc., such products for sale, commercial use,licensing, etc.

[0006] Kinases

[0007] KSE 336 is a protein kinase, exhibiting, e.g., a serine/threonineactivity. Protein kinases are a diverse and large group of enzymes thatcatalyze the transfer of a phosphate group. In most cases, the gammaphosphate of ATP or GTP serves as the phosphate donor, and a proteinalcohol or phenol group acts as the phosphate acceptor. Protein kinasesare ubiquitous in eukaryotes, playing an important role in development,differentiation, cell division, cell function, and signaling pathways.Kinases can be divided into different groups based on sequence homologyand function. These groups include: (1) CMGC, (2) PTK, (3) STE, (4)Gcyc, (5) AGC, (6) CAMK, and (7) CK1. Thorner et al., Cell Signaling,Chapter 2, Pages 8-9, New Science Press.

[0008] (1) The CMGC kinases includes cyclin-dependent kinases (Cdks),MAPKs, glycogen synthase kinases (GSKs), and CTD kinases. These enzymesphosphorylate serines and threonines at -Ser-Pro- or -Tbr-Pro-.

[0009] (2) PTK kinases are the tyrosine kinases. These include receptorkinases having a ligand binding extracellular domain and anintracellular kinase domain, as well as intracellularly expressed PTKs.

[0010] (3) The STE group includes homologs of yeast Ste20 (PAK), Ste11(MAPKKK), and Ste7 (MAPKK). These are serine/threonine kinases, some ofwhich have dual specificity. A major group of the STE kinases are thekinases involved in the mitogen-activated protein kinase (MAPK) cascade.MAPK cascades play key roles in relaying various physiological,environmental, or pathological signals from the environment to thetranscriptional machinery in the nucleus. MAPK is activated by dualphosphorylation of threonine and tyrosine residues in a TXY motiflocated between subdomains VII and VIII of the kinase catalytic domainby MAPK kinase (MAPKK). MAPKK is, in turn, activated by MAPKK kinase(MAPKKK). The general path of the cascade can therefore be characterizedas: Stimulus→MAPKKK→MAPKK→MAPK→Response. In S. cerevisiae, Ste20, Ste11,and Ste7 form a MAPK cascade which functions in the pheromone-inducedsignal transmission.

[0011] (4) The Gcyc group most closely resembles the kinase-like domainfound in guanylate cyclases. The specificity of these enzymes has notbeen completely characterized.

[0012] (5) The AGC group is made up the PKAs (cAMP-dependent proteinkinase), PTGs (cGMP-dependent kinase), and certain lipid activatedprotein kinases (protein kinase C or PKC). They phosphorylate serine orthreonine residues. These enzymes are comprised of multiple subunits.For example, PKA consists a catalytic (C) and a regulatory (R) subunit.A PKA can have multiple regulatory and/or catalytic subunits. Forinstance, mammalian 5′AMP-activated protein kinase (AMPK) comprises asingle catalytic alpha-subunit and two noncatalytic subunits, beta- andgamma-. There are multiple isoforms for each subunit. See, e.g.,Stapleton et al., J. Biol. Chem., 271:611-614, 1996.

[0013] (6) The CAMK group of protein kinases comprisecalciumi/calmodulin regulated, cAMP-regulated, and ELKL motif kinases.These enzymes phosphorylate serine and threonine residues.

[0014] (7) The CK1 group is so named because its family members resemblethe casein kinase 1. The function of this class has been difficult todocument, but they typically consist of a single catalytic subunitcapable of phosphorylating serine residues. Many different isoforms foreach type have been described.

[0015] KSE336 possesses serine and/or threonine kinase activity similarto the activity displayed by kinases in groups 1, 3, 5, 6, and 7. By itsamino acid sequence, it is most similar the AGC (5) group of kinases.

[0016] KSE336

[0017] KSE336 codes for a serine/threonine kinase (“STK”). Two forms ofit have been identified, KSE336-1 (FB1620G06) and KSE336-2 (AB1138D11).KSE336-1 is 668 amino acids, and KSE336-2 is 585 amino acids. Nucleotideand corresponding amino acid sequences of KSE336-1 are shown in SEQ IDNOS. 1 and 2, and SEQ ID NOS 3 and 4 for KSE336-2. The serine threoninekinase domain is found at amino acid positions 19-270 in KSE336-1, and1-185 in KSE336-2. A protein kinase active-site signature is found atamino acid positions 137-149 in KSE336-1 and is present in KSE336-2, aswell. See, FIG. 2. The two forms differ from each other only at the 5′end. See, FIG. 2. The open reading frames differ by only 4 base pairs.Alignment with genomic DNA reveals that this difference is derived fromthe exon-intron splicing site as follows:

[0018] Intron 5′ donor sequence

[0019] Intron 3′ accepter sequence _(—————)GTAGGT--------------------------------------------CAG_(——————)      1   2

[0020] The KSE336-1 cDNA was derived from a transcript that was splicedusing the first GT as the 5′ donor site, while KSE336-2 was splicedusing the second GT as the 5′ donor site. As a result, KSE336-2 has 4more base pairs than KSE336-1. Since this difference occurs in codingsequence, a shift in the open reading frame was observed, as reflectedin the different 5′ ends. Polymorphisms are shown in Table 2.

[0021] KSE336 maps to chromosomal band 11p15.5-pter (physical map from0.679 to 0.950 Mb; NT_(—)024164.2; AC074189; BAC clone, RP11-371C18).Partial clones, AF020089 and AJ006701, have been identified. See, FIG. 2for alignment. The present invention relates to fragments comprisingoverlapping regions, non-overlapping regions, regions comprisingvariations, etc., between the different forms of KSE336, and anyhomologs, truncated versions, polymorphisms, etc. Examples of sequencesrelated to KSE36 are shown in FIG. 2. Additional examples are describedbelow. As an illustration, but not to limit the invention in anyway, thepresent invention relates to such fragments as, amino acid positions1-71 (SEQ ID NOS 5 and 6) of KSE336-1; amino acid positions 72-668 ofKSE336-1; amino acid positions 1-8 of KSE336-2; amino acid positions647-659 of KSE336-1; 640-644 of KSE-1, 660-668 of KSE336-1, etc. Suchfragments can comprise, consist of, or consist essentially of, thesesequences.

[0022] The polypeptide coded for by KSE336 exhibits sequence identity toother STKs. It is related to kinases from other species are AF240782(mouse) and AF316542 (Drosophila). It also shares sequence homology withthe catalytic subunits of mammalian 5′AMP-activated protein kinase(AMPK) and yeast SNF1. The SNF1 family of PKAs are involved in glucosemetabolism. See, e.g., da Silva Xavier et al., Proc. Natl. Acad. Sci.,97:4023-4028, 2000. Mammalian AMPKs appear to be involved in regulatingthe response to nutritional stress, e.g., when ATP levels are low. See,e.g., Stapleton et al., J. Biol. Chem., 271:611-614, 1996.

[0023] KSE336 is also homologous to HrPOPK-1, an STK whose mRNA isdetected in early ascidian embryos. Sasakura et al., Mech. Dev.,76:161-163, 1998. Sequence homology is also observed between KSE336 andanother STK, SAD-1, a polypeptide which regulates presynaptic vesicleclustering and axon termination in C. elegans. See, e.g., Neuron,29(1):115-129, 2001.

[0024] KSE336 is predicted to have 19 exons with the followingstructure:    2 . . 320 (NT_024164)   321 . . 413 (AC074189,RP11-371C18) reverse orientation   414 . . 504 (NT_024164)   505 . . 641(NT_024164)   642 . . 758 (AC074189)   759 . . 792 (AC074189)   793 . .861 (AC091196,RP11-371C18)   862 . . 1008 (AC091196) 1009 . .1041(NT_024164) 1042 . . 1206(NT_024124) 1207 . . 1304(NT_024124) 1305 .. 1455(NT_024124) 1456 . . 1516(NT_024124) 1517 . . 1724(NT_024124) 1725. . 1773(NT_024124) 1774 . . 1897(NT_024124) 1898 . . 2078(NT_024124)2079 . . 2168(NT_024124) 2169 . . 2263 (NT_024124)

[0025] Expression

[0026] As show in FIG. 1, expression of KSE336 is restricted to thebrain and pancreas. The coincidence of brain and pancreas expression isespecially interesting since these cell types utilize common signalingpathways during development. Components of the Notch signaling pathwayare expressed during both neuronal and pancreatic cell differentiation(Apelqvist et al., Nature, 400:877-881, 1999; Jensen et al., NatureGenet., 24:36-44, 2000). Furthermore, embryonic stem (ES) cells whichdisplay neuronal cell markers have been induced to differentiate intoinsulin-producing pancreatic islet cells, indicating a closerelationship between the two cell types (Lumelsky et al., Science,292:1389-1394, 2001). Additionally, both tissues are exquisitelysensitive to changes in glucose and ATP levels, a function ofcAMP-dependent STKs, such as SNF1 and AMPK. KSE336 is also expressed inneural stem cells.

[0027] Disease Association

[0028] As indicated by its expression profile, KSE336 has a functionalrole in brain and pancreas. When the normal function of a gene isperturbed, the cells and tissues in which it is expressed arecorrespondingly affected, generally in a deleterious way. A range ofdifferent phenotypes are commonly observed, depending on the nature ofthe gene mutation and its interaction with other genetic andenvironmental factors. The brain and pancreas phenotypes associated withKSE336 aberrations, include, but are not limited to, e.g., astrocytoma,meningioma, pancreatic adenocarcinoma, insulin-dependent diabetesmellitus 2 (IDDM2), helicoid peripapillary chorioretinal degeneration(also known as atrophia areata), Beckwith-Wiedemann syndrome (see, e.g.,Hoovers et al., Proc. Natl. Acad. Sci., 92:12456-12460, 1995), andcongenital hyperinsulinism (e.g., Fournet et al., Horm. Res., 53:Suppl.1:2-6, 2000).

[0029] In addition to the above-mentioned disorders, KSE336 may beassociated with other conditions, e.g., which result from its expressionin tissues other than brain or pancreas. Such disorders include, but arenot limited to, arthrogryposis multiplex congenital distal type 2B(AMCD2B; Paris et al., Genomics, 69(2):196-202, 2000), Wilms Tumor2(WT2; see, e.g., U.S. Pat. No. 5,726,288).

[0030] The chromosomal region in which the KSE336 gene is located isinvolved in genomic imprinting, the phenomenon in which epigeneticmodification of a specific parental chromosome in the gamete or zygoteleads to monoallelic or differential expression of the two alleles ofthe gene in the offspring's somatic cells. An example of a diseaselocalized to 11p15 and implicated in defective genomic imprinting is theBeckwith-Wiedemann syndrome (BWS). BWS is a disorder of prenatalovergrowth, cancer, and hypoglycemia (associated with pancreatic islethyperplasia). It is known to be transmitted as an autosomal dominanttrait, but also occasionally arises spontaneously. Two separate domainsof imprinted genes appear to be involved in BWS. See, e.g., Lee et al.,Proc. Natl. Acad. Sci., 96:5203-5208, 1999; Maher and Reik, J. Clin.Invest., 105:247-252, 2000; Feinberg, J. Clin. Invest., 106:739-740,2000. About half of patients with BWS showed loss of imprinting (LOI)with LIT1, but only 20% with IGF2 (Lee et al., Proc. Natl. Acad. Sci.,96:5203-5208, 1999). Accordingly, nucleic acids of the presentinvention, including SNPs and other polymorphisms of it, can be use asprobes to analyze whether a gene has been imprinted.

[0031] Activity

[0032] By the phrase “serine/threonine kinase activity,” it is meant acatalytic activity in which a gamma phosphate from adenosinetriphosphate (ATP) is transferred to a serine or threonine residue in aprotein substrate. More generally, a “kinase activity” refers to theability of an enzyme to catalyze the transfer of a phosphate from onemolecule to another.

[0033] Kinase activity of KSE336, and biologically active fragmentsthereof, can be determined routinely using conventional assay methods.Kinase assays typically comprise the kinase enzyme, substrates, buffers,and components of a detection system. A typical kinase assay involves areaction of a protein kinase sample with a peptide substrate and agamma-labeled ATP, such as ³²P-ATP. The resulting labeled phosphoproteinis then separated from the gamma-labeled ATP. Separation and detectionof the phosphoprotein can be achieved through any suitable method. Whena radioactive label is utilized, the labeled phosphoprotein can beseparated from the unreacted gamma-³²P-ATP using an affinity membrane orgel electrophoresis, and then visualized on the gel usingautoradiography.

[0034] Non-radioactive methods can also be used. Methods can utilize anantibody which recognizes the phosphorylated substrate, e.g., ananti-phosphoserine or anti-phosphothreonine antibody. For instance,kinase enzyme can incubated with a substrate in the presence of ATP andkinase buffer under conditions which are effective for the enzyme tophosphorylate the substrate. The reaction mixture can be separated,e.g., electrophoretically, and then phosphorylation of the substrate canbe measured by Western blotting using an anti-phosphoserine oranti-phosphothreonine antibody. The antibody can be labeled with adetectable label, e.g., an enzyme, such as HRP, avidin or biotin,chemiluminescent reagents, etc. Other methods can utilize ELISA formats,affinity membrane separation, fluorescence polarization assays,luminescent assays, etc. Kinase assays are available commercially, e.g.,Cell Signaling Corporation (e.g., p44/42 MAP Kinase Assay Kit), AUSAUniversal Protein Kinase Assay Kit, ProMega (e.g., PepTag assays),SpinZyme colorimetric assays from Pierce, Calbiochem's ELISA-basedkinase assays, Upstate Biotechnology's ELISA-based kits usingchemiluminescent DuoLuX substrate from Vector Laboratories, PanVera'sfluorescent polarization kits, etc.

[0035] For kinase assays, see also, e.g., Kemp et al., “Design and useof peptide substrates for protein kinases,” Methods in Enzymol.,200:121-34, 1991; Wang et al., “Identification of the major site of ratprolactin phosphorylation as serine 177,” J. Biol. Chem., 271:2462-9,1996; Yasuda et al., “A synthetic peptide substrate for selective assayof protein kinase C,” Biochem. Biophys. Res. Comm., 166:1220-7, 1990;Gonzalez et al., “Use of the synthetic peptide neurogranin(28-43) as aselective protein kinase C substrate in assays of tissue homogenates,”Anal. Biochem., 215:184-9, 1993; Parker et al., “Development of highthroughput screening assays using fluorescence polarization: nuclearreceptor-ligand-binding and kinase/phosphatase assays,” J. Biomol.Screen., 5:77-88, April 2000. See, also., U.S. Pat. Nos. 6,203,994,6,074,861, 6,066,462, 6,004,757, and 5,741,689.

[0036] When a serine/threonine kinase activity is to be detected, asuitable substrate comprises serine and threonine residues, e.g., Elk-1,MBP, histones, such as H3, protamine, protamine sulfate, neurogranin,glycogen synthase, and fragments and fusion proteins thereof,HMRSAMSGLHLVKRR (SEQ ID NO 15), LRRASLG (SEQ ID NO 16), etc. Originally,a consensus PKC phosphorylation motif was determined to be RXXS/TXRX,where X indicates any amino acid. Generally, PKCs prefer basic residuesat positions −6, −4 and −2 to the Ser/Thr. cPKCs also preferred basicresidues at +2, +3 and +4, whereas nPKC and aPKCs preferred hydrophobicresidues at these positions. PKCmu deviates from this specificity,having an optimal motif which differs from other PKCs, with a strongselectivity for Leu at the −5 position. See, e.g., Toker, Frontiers inBioscience, 3:d1134-1147, 1998. PKAs can be assayed according to, e.g.,Davies et al., Eur. J. Biochem., 186:123-128, 1989; Roskoski, MethodsEnymol., 99:3-6, 1983; Cob and Corbin, Methods Enzymol., 159:202-208,1988. Consensus sequences for KSE336 can be determined analogously.

[0037] Nucleic Acids

[0038] A mammalian polynucleotide, or fragment thereof, of the presentinvention is a polynucleotide having a nucleotide sequence obtainablefrom a natural source. It therefore includes naturally-occurring normal,naturally-occurring mutant, and naturally-occurring polymorphic alleles(e.g., SNPs), differentially-spliced transcripts, splice-variants, etc.By the term “naturally-occurring,” it is meant that the polynucleotideis obtainable from a natural source, e.g., animal tissue and cells, bodyfluids, tissue culture cells, forensic samples. Natural sources include,e.g., living cells obtained from tissues and whole organisms, tumors,cultured cell lines, including primary and immortalized cell lines.Naturally-occurring mutations can include deletions (e.g., a truncatedamino- or carboxy-terminus), substitutions, inversions, or additions ofnucleotide sequence. These genes can be detected and isolated bypolynucleotide hybridization according to methods which one skilled inthe art would know, e.g., as discussed below.

[0039] A polynucleotide according to the present invention can beobtained from a variety of different sources. It can be obtained fromDNA or RNA, such as polyadenylated mRNA or total RNA, e.g., isolatedfrom tissues, cells, or whole organism. The polynucleotide can beobtained directly from DNA or RNA, from a cDNA library, from a genomiclibrary, etc. The polynucleotide can be obtained from a cell or tissue(e.g., from an embryonic or adult tissues) at a particular stage ofdevelopment, having a desired genotype, phenotype, disease status, etc.A polynucleotide which “codes without interruption” refers to apolynucleotide having a continuous open reading frame (“ORF”) ascompared to an ORF which is interrupted by introns or other noncodingsequences.

[0040] Polynucleotides and polypeptides (including any part of KSE336)can be excluded as compositions from the present invention if, e.g.,listed in a publicly available databases on the day this application wasfiled and/or disclosed in a patent application having an earlier filingor priority date than this application and/or conceived and/or reducedto practice earlier than a polynucleotide in this application. AJ006701(SEQ ID NO 17) and AF020089 (SEQ ID NO 18) can be excluded from thepresent invention, e.g., KSE336, fragments thereof, wherein suchfragment is not AJ006701 or AF020089.

[0041] As described herein, the phrase “an isolated polynucleotide whichis SEQ ID NO,” or “an isolated polynucleotide which is selected from SEQID NO,” refers to an isolated nucleic acid molecule from which therecited sequence was derived (e.g., a cDNA derived from mRNA; cDNAderived from genomic DNA). Because of sequencing errors, typographicalerrors, etc., the actual naturally-occurring sequence may differ from aSEQ ID listed herein. Thus, the phrase indicates the specific moleculefrom which the sequence was derived, rather than a molecule having thatexact recited nucleotide sequence, analogously to how a culturedepository number refers to a specific cloned fragment in a cryotube.

[0042] As explained in more detail below, a polynucleotide sequence ofthe invention can contain the complete sequence as shown in SEQ ID NO 1and 2, degenerate sequences thereof, anti-sense, muteins thereof, genescomprising said sequences, full-length cDNAs comprising said sequences,complete genomic sequences, fragments thereof (e.g., SEQ ID NOS 3 and4), homologs, primers, nucleic acid molecules which hybridize thereto,derivatives thereof, etc.

[0043] Genomic

[0044] The present invention also relates genomic DNA from which thepolynucleotides of the present invention can be derived. A genomic DNAcoding for a human, mouse, or other mammalian polynucleotide, can beobtained routinely, for example, by screening a genomic library (e.g., aYAC library) with a polynucleotide of the present invention, or bysearching nucleotide databases, such as GenBank and EMBL, for matches.Promoter and other regulatory regions can be identified upstream ofcoding and expressed RNAs, and assayed routinely for activity, e.g., byjoining to a reporter gene (e.g., CAT, GFP, alkaline phosphatase,luciferase, galatosidase). A promoter obtained from a brain and pancreasselective gene can be used, e.g., in gene therapy to obtaintissue-specific expression of a heterologous gene (e.g., coding for atherapeutic product or cytotoxin). Specific genomic promoter sequencesare listed in Table 1.

[0045] Constructs

[0046] A polynucleotide of the present invention can comprise additionalpolynucleotide sequences, e.g., sequences to enhance expression,detection, uptake, cataloging, tagging, etc. A polynucleotide caninclude only coding sequence; a coding sequence and additionalnon-naturally occurring or heterologous coding sequence (e.g., sequencescoding for leader, signal, secretory, targeting, enzymatic, fluorescent,antibiotic resistance, and other functional or diagnostic peptides);coding sequences and non-coding sequences, e.g., untranslated sequencesat either a 5′ or 3′ end, or dispersed in the coding sequence, e.g.,introns.

[0047] A polynucleotide according to the present invention also cancomprise an expression control sequence operably linked to apolynucleotide as described above. The phrase “expression controlsequence” means a polynucleotide sequence that regulates expression of apolypeptide coded for by a polynucleotide to which it is functionally(“operably”) linked. Expression can be regulated at the level of themRNA or polypeptide. Thus, the expression control sequence includesmRNA-related elements and protein-related elements. Such elementsinclude promoters, enhancers (viral or cellular), ribosome bindingsequences, transcriptional terminators, etc. An expression controlsequence is operably linked to a nucleotide coding sequence when theexpression control sequence is positioned in such a manner to effect orachieve expression of the coding sequence. For example, when a promoteris operably linked 5′ to a coding sequence, expression of the codingsequence is driven by the promoter. Expression control sequences caninclude an initiation codon and additional nucleotides to place apartial nucleotide sequence of the present invention in-frame in orderto produce a polypeptide (e.g., pET vectors from Promega have beendesigned to permit a molecule to be inserted into all three readingframes to identify the one that results in polypeptide expression).Expression control sequences can be heterologous or endogenous to thenormal gene.

[0048] A polynucleotide of the present invention can also comprisenucleic acid vector sequences, e.g., for cloning, expression,amplification, selection, etc. Any effective vector can be used. Avector is, e.g., a polynucleotide molecule which can replicateautonomously in a host cell, e.g., containing an origin of replication.Vectors can be useful to perform manipulations, to propagate, and/orobtain large quantities of the recombinant molecule in a desired host. Askilled worker can select a vector depending on the purpose desired,e.g., to propagate the recombinant molecule in bacteria, yeast, insect,or mammalian cells. The following vectors are provided by way ofexample. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10,Phagescript, phiX174, pBK Phagemid, pNH8A, pNH16a, pNH18Z, pNH46A(Stratagene); Bluescript KS+II (Stratagene); ptrc99a, pKK223-3,pKK233-3, pDR54 0, pRIT5 (Pharmacia). Eukaryotic: PWLNEO, pSV2CAT,pOG44, pXTI, pSG (Stratagene), pSVK3, PBPV, PMSG, pSVL (Pharmacia),pCR2.1/TOPO, pCRII/TOPO, pCR4/TOPO, pTrcHisB, pCMV6-XL4, etc. However,any other vector, e.g., plasmids, viruses, or parts thereof, may be usedas long as they are replicable and viable in the desired host. Thevector can also comprise sequences which enable it to replicate in thehost whose genome is to be modified.

[0049] Hybridization

[0050] Polynucleotide hybridization, as discussed in more detail below,is useful in a variety of applications, including, in gene detectionmethods, for identifying mutations, for making mutations, to identifyhomologs in the same and different species, to identify related membersof the same gene family, in diagnostic and prognostic assays, intherapeutic applications (e.g., where an antisense polynucleotide isused to inhibit expression), etc.

[0051] The ability of two single-stranded polynucleotide preparations tohybridize together is a measure of their nucleotide sequencecomplementarity, e.g., base-pairing between nucleotides, such as A-T,G-C, etc. The invention thus also relates to polynucleotides, and theircomplements, which hybridize to a polynucleotide comprising a nucleotidesequence as set forth in SEQ ID NO 1-6 and genomic sequences thereof. Anucleotide sequence hybridizing to the latter sequence will have acomplementary polynucleotide strand, or act as a template for one in thepresence of a polymerase (i.e., an appropriate polynucleotidesynthesizing enzyme). The present invention includes both strands ofpolynucleotide, e.g., a sense strand and an anti-sense strand.

[0052] Hybridization conditions can be chosen to select polynucleotideswhich have a desired amount of nucleotide complementarity with thenucleotide sequences set forth in SEQ ID NO 1-6 and genomic sequencesthereof. A polynucleotide capable of hybridizing to such sequence,preferably, possesses, e.g., about 70%, 75%, 80%, 85%, 87%, 90%, 92%,95%, 97%, 99%, or 100% complementarity, between the sequences. Thepresent invention particularly relates to polynucleotide sequences whichhybridize to the nucleotide sequences set forth in SEQ ID NO 1-6 orgenomic sequences thereof, under low or high stringency conditions.These conditions can be used, e.g., to select corresponding homologs innon-human species.

[0053] Polynucleotides which hybridize to polynucleotides of the presentinvention can be selected in various ways. Filter-type blots (i.e.,matrices containing polynucleotide, such as nitrocellulose), glasschips, and other matrices and substrates comprising polynucleotides(short or long) of interest, can be incubated in a prehybridizationsolution (e.g., 6×SSC, 0.5% SDS, 100 μg/ml denatured salmon sperm DNA,5× Denhardt's solution, and 50% formamide), at 22-68° C., overnight, andthen hybridized with a detectable polynucleotide probe under conditionsappropriate to achieve the desired stringency. In general, when highhomology or sequence identity is desired, a high temperature can be used(e.g., 65° C.). As the homology drops, lower washing temperatures areused. For salt concentrations, the lower the salt concentration, thehigher the stringency. The length of the probe is another consideration.Very short probes (e.g., less than 100 base pairs) are washed at lowertemperatures, even if the homology is high. With short probes, formamidecan be omitted. See, e.g., Current Protocols in Molecular Biology,Chapter 6, Screening of Recombinant Libraries; Sambrook et al.,Molecular Cloning, 1989, Chapter 9.

[0054] For instance, high stringency conditions can be achieved byincubating the blot overnight (e.g., at least 12 hours) with a longpolynucleotide probe in a hybridization solution containing, e.g., about5×SSC, 0.5% SDS, 100 μg/ml denatured salmon sperm DNA and 50% formamide,at 42° C. Blots can be washed at high stringency conditions that allow,e.g., for less than 5% bp mismatch (e.g., wash twice in 0.1% SSC and0.1% SDS for 30 min at 65° C.), i.e., selecting sequences having 95% orgreater sequence identity.

[0055] Other non-limiting examples of high stringency conditionsincludes a final wash at 65° C. in aqueous buffer containing 30 mM NaCland 0.5% SDS. Another example of high stringent conditions ishybridization in 7% SDS, 0.5 M NaPO₄, pH 7, 1 mM EDTA at 50° C., e.g.,overnight, followed by one or more washes with a 1% SDS solution at 42°C. Whereas high stringency washes can allow for less than 5% mismatch,reduced or low stringency conditions can permit up to 20% nucleotidemismatch. Hybridization at low stringency can be accomplished as above,but using lower formamide conditions, lower temperatures and/or lowersalt concentrations, as well as longer periods of incubation time.

[0056] Hybridization can also be based on a calculation of meltingtemperature (Tm) of the hybrid formed between the probe and its target,as described in Sambrook et al. Generally, the temperature Tm at which ashort oligonucleotide (containing 18 nucleotides or fewer) will meltfrom its target sequence is given by the following equation: Tm=(numberof A's and T's)×2° C.+(number of C's and G's)×4° C. For longermolecules, Tm=81.5+16.6 log₁₀[Na+]+0.41(% GC)−600/N where [Na+] is themolar concentration of sodium ions, % GC is the percentage of GC basepairs in the probe, and N is the length. Hybridization can be carriedout at several degrees below this temperature to ensure that the probeand target can hybridize. Mismatches can be allowed for by lowering thetemperature even further.

[0057] Stringent conditions can be selected to isolate sequences, andtheir complements, which have, e.g., at least about 90%, 95%, or 97%,nucleotide complementarity between the probe (e.g., a shortpolynucleotide of SEQ ID NO 1-6 or genomic sequences thereof) and atarget polynucleotide.

[0058] Other homologs of polynucleotides of the present invention can beobtained from mammalian and non-mammalian sources according to variousmethods. For example, hybridization with a polynucleotide can beemployed to select homologs, e.g., as described in Sambrook et al.,Molecular Cloning, Chapter 11, 1989. Such homologs can have varyingamounts of nucleotide and amino acid sequence identity and similarity tosuch polynucleotides of the present invention. Mammalian organismsinclude, e.g., mice, rats, monkeys, pigs, cows, etc. Non-mammalianorganisms include, e.g., vertebrates, invertebrates, zebra fish,chicken, Drosophila, C. elegans, Xenopus, yeast such as S. pombe, S.cerevisiae, roundworms, prokaryotes, plants, Arabidopsis, artemia,viruses, etc. The degree of nucleotide sequence identity between humanand mouse can be about, e.g. 70% or more, 85% or more for open readingframes, etc.

[0059] Alignment

[0060] Alignments can be accomplished by using any effective algorithm.For pairwise alignments of DNA sequences, the methods described byWilbur-Lipman (e.g., Wilbur and Lipman, Proc. Natl. Acad. Sci.,80:726-730, 1983) or Martinez/Needleman-Wunsch (e.g., Martinez, NucleicAcid Res., 11:4629-4634, 1983) can be used. For instance, if theMartinez/Needleman-Wunsch DNA alignment is applied, the minimum matchcan be set at 9, gap penalty at 1.10, and gap length penalty at 0.33.The results can be calculated as a similarity index, equal to the sum ofthe matching residues divided by the sum of all residues and gapcharacters, and then multiplied by 100 to express as a percent.Similarity index for related genes at the nucleotide level in accordancewith the present invention can be greater than 70%, 80%, 85%, 90%, 95%,99%, or more. Pairs of protein sequences can be aligned by theLipman-Pearson method (e.g., Lipman and Pearson, Science, 227:1435-1441,1985) with k-tuple set at 2, gap penalty set at 4, and gap lengthpenalty set at 12. Results can be expressed as percent similarity index,where related genes at the amino acid level in accordance with thepresent invention can be greater than 65%, 70%, 75%, 80%, 85%, 90%, 95%,99%, or more. Various commercial and free sources of alignment programsare available, e.g., MegAlign by DNA Star, BLAST (National Center forBiotechnology Information), BCM (Baylor College of Medicine) Launcher,etc.

[0061] Percent sequence identity can also be determined by otherconventional methods, e.g., as described in Altschul et al., Bull. Math.Bio. 48: 603-616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci.USA 89:10915-10919, 1992.

[0062] Specific Polynucleotide Probes

[0063] A polynucleotide of the present invention can comprise anycontinuous nucleotide sequence of SEQ ID NO 1-6, sequences which sharesequence identity thereto, or complements thereof. The term “probe”refers to any substance that can be used to detect, identify, isolate,etc., another substance. A polynucleotide probe is comprised of nucleicacid can be used to detect, identify, etc., other nucleic acids, such asDNA and RNA.

[0064] These polynucleotides can be of any desired size that iseffective to achieve the specificity desired. For example, a probe canbe from about 7 or 8 nucleotides to several thousand nucleotides,depending upon its use and purpose. For instance, a probe used as aprimer PCR can be shorter than a probe used in an ordered array ofpolynucleotide probes. Probe sizes vary, and the invention is notlimited in any way by their size, e.g., probes can be from about 7-2000nucleotides, 7-1000, 8-700, 8-600, 8-500, 8-400, 8-300, 8-150, 8-100,8-75, 7-50, 10-25, 14-16, at least about 8, at least about 10, at leastabout 15, at least about 25, etc. The polynucleotides can havenon-naturally-occurring nucleotides, e.g., inosine, AZT, 3TC, etc. Thepolynucleotides can have 100% sequence identity or complementarity to asequence of SEQ ID NO 1-6, or it can have mismatches or nucleotidesubstitutions, e.g., 1, 2, 3, 4, or 5 substitutions. The probes can besingle-stranded or double-stranded.

[0065] In accordance with the present invention, a polynucleotide can bepresent in a kit, where the kit includes, e.g., one or morepolynucleotides, a desired buffer (e.g., phosphate, tris, etc.),detection compositions, RNA or cDNA from different tissues to be used ascontrols, libraries, etc. The polynucleotide can be labeled orunlabeled, with radioactive or non-radioactive labels as known in theart. Kits can comprise one or more pairs of polynucleotides foramplifying nucleic acids specific for KSE336, e.g., comprising a forwardand reverse primer effective in PCR. These include both sense andanti-sense orientations. For instance, in PCR-based methods (such asRT-PCR), a pair of primers are typically used, one having a sensesequence and the other having an antisense sequence.

[0066] Another aspect of the present invention is a nucleotide sequencethat is specific to, or for, a selective polynucleotide. The phrases“specific for” or “specific to” a polynucleotide have a functionalmeaning that the polynucleotide can be used to identify the presence ofone or more target genes in a sample. It is specific in the sense thatit can be used to detect polynucleotides above background noise(“non-specific binding”). A specific sequence is a defined order ofnucleotides which occurs in the polynucleotide, e.g., in the nucleotidesequences of SEQ ID NO 1-6. A probe or mixture of probes can comprise asequence or sequences that are specific to a plurality of targetsequences, e.g., where the sequence is a consensus sequence, afunctional domain, etc., e.g., capable of recognizing a family ofrelated genes. Such sequences can be used as probes in any of themethods described herein or incorporated by reference. Both sense andantisense nucleotide sequences are included. A specific polynucleotideaccording to the present invention can be determined routinely.

[0067] A polynucleotide comprising a specific sequence can be used as ahybridization probe to identify the presence of, e.g., human or mousepolynucleotide, in a sample comprising a mixture of polynucleotides,e.g., on a Northern blot. Hybridization can be performed under highstringent conditions (see, above) to select polynucleotides (and theircomplements which can contain the coding sequence) having at least 90%,95%, 99%, etc., identity (i.e., complementarity) to the probe, but lessstringent conditions can also be used. A specific polynucleotidesequence can also be fused in-frame, at either its 5′ or 3′ end, tovarious nucleotide sequences as mentioned throughout the patent,including coding sequences for enzymes, detectable markers, GFP, etc,expression control sequences, etc.

[0068] A polynucleotide probe, especially one that is specific to apolynucleotide of the present invention, can be used in gene detectionand hybridization methods as already described. In one embodiment, aspecific polynucleotide probe can be used to detect whether a particulartissue or cell-type is present in a target sample. To carry out such amethod, a selective polynucleotide can be chosen which is characteristicof the desired target tissue. Such polynucleotide is preferably chosenso that it is expressed or displayed in the target tissue, but not inother tissues which are present in the sample. For instance, ifdetection of brain and pancreas is desired, it may not matter whetherthe selective polynucleotide is expressed in other tissues, as long asit is not expressed in cells normally present in blood, e.g., peripheralblood mononuclear cells. Starting from the selective polynucleotide, aspecific polynucleotide probe can be designed which hybridizes (ifhybridization is the basis of the assay) under the hybridizationconditions to the selective polynucleotide, whereby the presence of theselective polynucleotide can be determined.

[0069] Probes which are specific for polynucleotides of the presentinvention can also be prepared using involve transcription-basedsystems, e.g., incorporating an RNA polymerase promoter into a selectivepolynucleotide of the present invention, and then transcribinganti-sense RNA using the polynucleotide as a template. See, e.g., U.S.Pat. No. 5,545,522.

[0070] Polynucleotide Composition

[0071] A polynucleotide according to the present invention can comprise,e.g., DNA, RNA, synthetic polynucleotide, peptide polynucleotide,modified nucleotides, dsDNA, ssDNA, ssRNA, dsRNA, and mixtures thereof.A polynucleotide can be single- or double-stranded, triplex, DNA:RNA,duplexes, comprise hairpins, and other secondary structures, etc.Nucleotides comprising a polynucleotide can be joined via various knownlinkages, e.g., ester, sulfamate, sulfamide, phosphorothioate,phosphoramidate, methylphosphonate, carbamate, etc., depending on thedesired purpose, e.g., resistance to nucleases, such as RNAse H,improved in vivo stability, etc. See, e.g., U.S. Pat. No. 5,378,825. Anydesired nucleotide or nucleotide analog can be incorporated, e.g.,6-mercaptoguanine, 8-oxo-guanine, etc.

[0072] Various modifications can be made to the polynucleotides, such asattaching detectable markers (avidin, biotin, radioactive elements,fluorescent tags and dyes, energy transfer labels, energy-emittinglabels, binding partners, etc.) or moieties which improve hybridization,detection, and/or stability. The polynucleotides can also be attached tosolid supports, e.g., nitrocellulose, magnetic or paramagneticmicrospheres (e.g., as described in U.S. Pat. Nos. 5,411,863; 5,543,289;for instance, comprising ferromagnetic, supermagnetic, paramagnetic,superparamagnetic, iron oxide and polysaccharide), nylon, agarose,diazotized cellulose, latex solid microspheres, polyacrylamides, etc.,according to a desired method. See, e.g., U.S. Pat. Nos. 5,470,967,5,476,925, and 5,478,893.

[0073] Polynucleotide according to the present invention can be labeledaccording to any desired method. The polynucleotide can be labeled usingradioactive tracers such as ³²P, ³⁵S, ³H, or ¹⁴C, to mention somecommonly used tracers. The radioactive labeling can be carried outaccording to any method, such as, for example, terminal labeling at the3′ or 5′ end using a radiolabeled nucleotide, polynucleotide kinase(with or without dephosphorylation with a phosphatase) or a ligase(depending on the end to be labeled). A non-radioactive labeling canalso be used, combining a polynucleotide of the present invention withresidues having immunological properties (antigens, haptens), a specificaffinity for certain reagents (ligands), properties enabling detectableenzyme reactions to be completed (enzymes or coenzymes, enzymesubstrates, or other substances involved in an enzymatic reaction), orcharacteristic physical properties, such as fluorescence or the emissionor absorption of light at a desired wavelength, etc.

[0074] Nucleic Acid Detection Methods

[0075] Another aspect of the present invention relates to methods andprocesses for detecting KSE336. Detection methods have a variety ofapplications, including for diagnostic, prognostic, forensic, andresearch applications. To accomplish gene detection, a polynucleotide inaccordance with the present invention can be used as a “probe.” The term“probe” or “polynucleotide probe” has its customary meaning in the art,e.g., a polynucleotide which is effective to identify (e.g., byhybridization), when used in an appropriate process, the presence of atarget polynucleotide to which it is designed. Identification caninvolve simply determining presence or absence, or it can bequantitative, e.g., in assessing amounts of a gene or gene transcriptpresent in a sample. Probes can be useful in a variety of ways, such asfor diagnostic purposes, to identify homologs, and to detect,quantitate, or isolate a polynucleotide of the present invention in atest sample.

[0076] Assays can be utilized which permit quantification and/orpresence/absence detection of a target nucleic acid in a sample. Assayscan be performed at the single-cell level, or in a sample comprisingmany cells, where the assay is “averaging” expression over the entirecollection of cells and tissue present in the sample. Any suitable assayformat can be used, including, but not limited to, e.g., Southern blotanalysis, Northern blot analysis, polymerase chain reaction (“PCR”)(e.g., Saiki et al., Science, 241:53, 1988; U.S. Pat. Nos. 4,683,195,4,683,202, and 6,040,166; PCR Protocols. A Guide to Methods andApplications, Innis et al., eds., Academic Press, New York, 1990),reverse transcriptase polymerase chain reaction (“RT-PCR”), anchoredPCR, rapid amplification of CDNA ends (“RACE”) (e.g., Schaefer in GeneCloning and Analysis. Current Innovations, Pages 99-115, 1997), ligasechain reaction (“LCR”) (EP 320 308), one-sided PCR (Ohara et al., Proc.Natl. Acad. Sci., 86:5673-5677, 1989), indexing methods (e.g., U.S. Pat.No. 5,508,169), in situ hybridization, differential display (e.g., Lianget al., Nucl. Acid. Res., 21:3269-3275, 1993; U.S. Pat. Nos. 5,262,311,5,599,672 and 5,965,409; WO97/18454; Prashar and Weissman, Proc. Natl.Acad. Sci., 93:659-663, and U.S. Pat. Nos. 6,010,850 and 5,712,126;Welsh et al., Nucleic Acid Res., 20:4965-4970, 1992, and U.S. Pat. No.5,487,985) and other RNA fingerprinting techniques, nucleic acidsequence based amplification (“NASBA”) and other transcription basedamplification systems (e.g., U.S. Pat. Nos. 5,409,818 and 5,554,527; WO88/10315), polynucleotide arrays (e.g., U.S. Pat. Nos. 5,143,854,5,424,186; 5,700,637, 5,874,219, and 6,054,270; PCT WO 92/10092; PCT WO90/15070), Qbeta Replicase (PCT/US87/00880), Strand DisplacementAmplification (“SDA”), Repair Chain Reaction (“RCR”), nucleaseprotection assays, subtraction-based methods, Rapid-Scan™, etc.Additional useful methods include, but are not limited to, e.g.,template-based amplification methods, competitive PCR (e.g., U.S. Pat.No. 5,747,251), redox-based assays (e.g., U.S. Pat. No. 5,871,918),Taqman-based assays (e.g., Holland et al., Proc. Natl. Acad, Sci.,88:7276-7280, 1991; U.S. Pat. Nos. 5,210,015 and 5,994,063), real-timefluorescence-based monitoring (e.g., U.S. Pat. No. 5,928,907), molecularenergy transfer labels (e.g., U.S. Pat. Nos. 5,348,853, 5,532,129,5,565,322, 6,030,787, and 6,117,635; Tyagi and Kramer, Nature Biotech.,14:303-309, 1996). Any method suitable for single cell analysis of geneor protein expression can be used, including in situ hybridization,immunocytochemistry, MACS, FACS, flow cytometry, etc. For single cellassays, expression products can be measured using antibodies, PCR, orother types of nucleic acid amplification (e.g., Brady et al., MethodsMol. & Cell. Biol. 2, 17-25, 1990; Eberwine et al., 1992, Proc. Natl.Acad. Sci., 89, 3010-3014, 1992; U.S. Pat. No. 5,723,290). These andother methods can be carried out conventionally, e.g., as described inthe mentioned publications.

[0077] Many of such methods may require that the polynucleotide islabeled, or comprises a particular nucleotide type useful for detection.The present invention includes such modified polynucleotides that arenecessary to carry out such methods. Thus, polynucleotides can be DNA,RNA, DNA:RNA hybrids, PNA, etc., and can comprise any modification orsubstituent which is effective to achieve detection.

[0078] Detection can be desirable for a variety of different purposes,including research, diagnostic, prognostic, and forensic. For diagnosticpurposes, it may be desirable to identify the presence or quantity of apolynucleotide sequence in a sample, where the sample is obtained fromtissue, cells, body fluids, etc. In a preferred method as described inmore detail below, the present invention relates to a method ofdetecting a polynucleotide comprising, contacting a targetpolynucleotide in a test sample with a polynucleotide probe underconditions effective to achieve hybridization between the target andprobe; and detecting hybridization.

[0079] Any test sample in which it is desired to identify apolynucleotide or polypeptide thereof can be used, including, e.g.,blood, urine, saliva, stool (for extracting nucleic acid, see, e.g.,U.S. Pat. No. 6,177,251), swabs comprising tissue, biopsied tissue,tissue sections, cultured cells, etc.

[0080] Detection can be accomplished in combination with polynucleotideprobes for other genes, e.g., genes which are expressed in other diseasestates, tissues, cells, such as brain, heart, kidney, spleen, thymus,liver, stomach, small intestine, colon, muscle, lung, testis, placenta,pituitary, thyroid, skin, adrenal gland, pancreas, salivary gland,uterus, ovary, prostate gland, peripheral blood cells (T-cells,lymphocytes, etc.), embryo, normal breast fat, adult and embryonic stemcells, specific cell-types, such as endothelial, epithelial, myocytes,adipose, luminal epithelial, basoepithelial, myoepithelial, stromalcells, etc.

[0081] Polynucleotides can be used in wide range of methods andcompositions, including for detecting, diagnosing, staging, grading,assessing, prognosticating, etc. diseases and disorders associated withKSE336, for monitoring or assessing therapeutic and/or preventativemeasures, in ordered arrays, etc. Any method of detecting genes andpolynucleotides of SEQ ID NO 1-6 can be used; certainly, the presentinvention is not to be limited how such methods are implemented.

[0082] Along these lines, the present invention relates to methods ofdetecting KSE336 in a sample comprising nucleic acid. Such methods cancomprise one or more the following steps in any effective order, e.g.,contacting said sample with a polynucleotide probe under conditionseffective for said probe to hybridize specifically to nucleic acid insaid sample, and detecting the presence or absence of probe hybridizedto nucleic acid in said sample, wherein said probe is a polynucleotidewhich is SEQ ID NO 1-6, a polynucleotide having, e.g., about 70%, 80%,85%, 90%, 95%, 99%, or more sequence identity thereto, effective orspecific fragments thereof, or complements thereto. The detection methodcan be applied to any sample, e.g., cultured primary, secondary, orestablished cell lines, tissue biopsy, blood, urine, stool, and otherbodily fluids, for any purpose.

[0083] Contacting the sample with probe can be carried out by anyeffective means in any effective environment. It can be accomplished ina solid, liquid, frozen, gaseous, amorphous, solidified, coagulated,colloid, etc., mixtures thereof, matrix. For instance, a probe in anaqueous medium can be contacted with a sample which is also in anaqueous medium, or which is affixed to a solid matrix, or vice-versa.

[0084] Generally, as used throughout the specification, the term“effective conditions” means, e.g., the particular milieu in which thedesired effect is achieved. Such a milieu, includes, e.g., appropriatebuffers, oxidizing agents, reducing agents, pH, co-factors, temperature,ion concentrations, suitable age and/or stage of cell (such as, inparticular part of the cell cycle, or at a particular stage whereparticular genes are being expressed) where cells are being used,culture conditions (including substrate, oxygen, carbon dioxide, etc.).When hybridization is the chosen means of achieving detection, the probeand sample can be combined such that the resulting conditions arefunctional for said probe to hybridize specifically to nucleic acid insaid sample.

[0085] The phrase “hybridize specifically” indicates that thehybridization between single-stranded polynucleotides is based onnucleotide sequence complementarity. The effective conditions areselected such that the probe hybridizes to a preselected and/or definitetarget nucleic acid in the sample. For instance, if detection of apolynucleotide set forth in SEQ ID NO 1-6 is desired, a probe can beselected which can hybridize to such target gene under high stringentconditions, without significant hybridization to other genes in thesample. To detect homologs of a polynucleotide set forth in SEQ ID NO 1and 2, the effective hybridization conditions can be less stringent,and/or the probe can comprise codon degeneracy, such that a homolog isdetected in the sample.

[0086] As already mentioned, the methods can be carried out by anyeffective process, e.g., by Northern blot analysis, polymerase chainreaction (PCR), reverse transcriptase PCR, RACE PCR, in situhybridization, etc., as indicated above. When PCR based techniques areused, two or more probes are generally used. One probe can be specificfor a defined sequence which is characteristic of a selectivepolynucleotide, but the other probe can be specific for the selectivepolynucleotide, or specific for a more general sequence, e.g., asequence such as polyA which is characteristic of mRNA, a sequence whichis specific for a promoter, ribosome binding site, or othertranscriptional features, a consensus sequence (e.g., representing afunctional domain). For the former aspects, 5′ and 3′ probes (e.g.,polyA, Kozak, etc.) are preferred which are capable of specificallyhybridizing to the ends of transcripts. When PCR is utilized, the probescan also be referred to as “primers” in that they can prime a DNApolymerase reaction.

[0087] In addition to testing for the presence or absence ofpolynucleotides, the present invention also relates to determining theamounts at which polynucleotides of the present invention are expressedin sample and determining the differential expression of suchpolynucleotides in samples. Such methods can involve substantially thesame steps as described above for presence/absence detection, e.g.,contacting with probe, hybridizing, and detecting hybridized probe, butusing more quantitative methods and/or comparisons to standards.

[0088] The amount of hybridization between the probe and target can bedetermined by any suitable methods, e.g., PCR, RT-PCR, RACE PCR,Northern blot, polynucleotide microarrays, Rapid-Scan, etc., andincludes both quantitative and qualitative measurements. For furtherdetails, see the hybridization methods described above and below.Determining by such hybridization whether the target is differentiallyexpressed (e.g., up-regulated or down-regulated) in the sample can alsobe accomplished by any effective means. For instance, the target'sexpression pattern in the sample can be compared to its pattern in aknown standard, such as in a normal tissue, or it can be compared toanother gene in the same sample. When a second sample is utilized forthe comparison, it can be a sample of normal tissue that is known not tocontain diseased cells. The comparison can be performed on samples whichcontain the same amount of RNA (such as polyadenylated RNA or totalRNA), or, on RNA extracted from the same amounts of starting tissue.Such a second sample can also be referred to as a control or standard.Hybridization can also be compared to a second target in the same tissuesample. Experiments can be performed that determine a ratio between thetarget nucleic acid and a second nucleic acid (a standard or control),e.g., in a normal tissue. When the ratio between the target and controlare substantially the same in a normal and sample, the sample isdetermined or diagnosed not to contain cells. However, if the ratio isdifferent between the normal and sample tissues, the sample isdetermined to contain cancer cells. The approaches can be combined, andone or more second samples, or second targets can be used. Any secondtarget nucleic acid can be used as a comparison, including“housekeeping” genes, such as beta-actin, alcohol dehydrogenase, or anyother gene whose expression does not vary depending upon the diseasestatus of the cell.

[0089] Methods of Identifying Polymorphisms, Mutations, etc., of KSE336

[0090] Polynucleotides of the present invention can also be utilized toidentify mutant alleles, SNPs, gene rearrangements and modifications,and other polymorphisms of the wild-type gene. Mutant alleles,polymorphisms, SNPs, etc., can be identified and isolated from cancersthat are known, or suspected to have, a genetic component.Identification of such genes can be carried out routinely (see, abovefor more guidance), e.g., using PCR, hybridization techniques, directsequencing, mismatch reactions (see, e.g., above), RFLP analysis, SSCP(e.g., Orita et al., Proc. Natl. Acad. Sci., 86:2766, 1992), etc., wherea polynucleotide having a sequence selected from SEQ ID NO 1 and 2 isused as a probe. The selected mutant alleles, SNPs, polymorphisms, etc.,can be used diagnostically to determine whether a subject has, or issusceptible to a disorder associated with KSE336, as well as to designtherapies and predict the outcome of the disorder. Methods involve,e.g., diagnosing a disorder associated with KSE336, comprising,detecting the presence of a mutation in a gene represented by apolynucleotide selected from SEQ ID NO 1 and 2. The detecting can becarried out by any effective method, e.g., obtaining cells from asubject, determining the gene sequence or structure of a target gene(using, e.g., mRNA, cDNA, genomic DNA, etc), comparing the sequence orstructure of the target gene to the structure of the normal gene,whereby a difference in sequence or structure indicates a mutation inthe gene in the subject. Polynucleotides can also be used to test formutations, SNPs, polymorphisms, etc., e.g., using mismatch DNA repairtechnology as described in U.S. Pat. No. 5,683,877; U.S. Pat. No.5,656,430; Wu et al., Proc. Natl. Acad. Sci., 89:8779-8783, 1992.

[0091] The present invention also relates to methods of detectingpolymorphisms in KSE336, comprising, e.g., comparing the structure of:genomic DNA comprising all or part of KSE336, mRNA comprising all orpart of KSE336, cDNA comprising all or part of KSE336, or a polypeptidecomprising all or part of KSE336, with the structure of KSE336 set forthin SEQ ID NO 1-6. The methods can be carried out on a sample from anysource, e.g., cells, tissues, body fluids, blood, urine, stool, hair,egg, sperm, etc.

[0092] These methods can be implemented in many different ways. Forexample, “comparing the structure” steps include, but are not limitedto, comparing restriction maps, nucleotide sequences, amino acidsequences, RFLPs, Dnase sites, DNA methylation fingerprints (e.g., U.S.Pat. No. 6,214,556), protein cleavage sites, molecular weights,electrophoretic mobilities, charges, ion mobility, etc., between astandard KSE336 and a test KSE336. The term “structure” can refer to anyphysical characteristics or configurations which can be used todistinguish between nucleic acids and polypeptides. The methods andinstruments used to accomplish the comparing step depends upon thephysical characteristics which are to be compared. Thus, varioustechniques are contemplated, including, e.g., sequencing machines (bothamino acid and polynucleotide), electrophoresis, mass spectrometer (U.S.Pat. Nos. 6,093,541, 6,002,127), liquid chromatography, HPLC, etc.

[0093] To carry out such methods, “all or part” of the gene orpolypeptide can be compared. For example, if nucleotide sequencing isutilized, the entire gene can be sequenced, including promoter, introns,and exons, or only parts of it can be sequenced and compared, e.g., exon1, exon 2, etc.

[0094] Mutagenesis

[0095] Mutated polynucleotide sequences of the present invention areuseful for various purposes, e.g., to create mutations of thepolypeptides they encode, to identify functional regions of genomic DNA,to produce probes for screening libraries, etc. Mutagenesis can becarried out routinely according to any effective method, e.g.,oligonucleotide-directed (Smith, M., Ann. Rev. Genet. 19:423-463, 1985),degenerate oligonucleotide-directed (Hill et al., Method Enzymology,155:558-568, 1987), region-specific (Myers et al., Science, 229:242-246,1985; Derbyshire et al., Gene, 46:145, 1986; Ner et al., DNA, 7:127,1988), linker-scanning (McKnight and Kingsbury, Science, 217:316-324,1982), directed using PCR, recursive ensemble mutagenesis (Arkin andYourvan, Proc. Natl. Acad. Sci., 89:7811-7815, 1992), random mutagenesis(e.g., U.S. Pat. Nos. 5,096,815; 5,198,346; and 5,223,409),site-directed mutagenesis (e.g., Walder et al., Gene, 42:133, 1986;Bauer et al., Gene, 37:73, 1985; Craik, Bio Techniques, Jan. 12-19,1985; Smith et al., Genetic Engineering: Principles and Methods, PlenumPress, 1981), phage display (e.g., Lowman et al., Biochem.30:10832-10837, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPOPublication WO 92/06204), etc. Desired sequences can also be produced bythe assembly of target sequences using mutually priming oligonucleotides(Uhlmann, Gene, 71:29-40, 1988). For directed mutagenesis methods,analysis of the three-dimensional structure of the KSE336 polypeptidecan be used to guide and facilitate making mutants which effectpolypeptide activity. Sites of substrate-enzyme interaction or otherbiological activities can also be determined by analysis of crystalstructure as determined by such techniques as nuclear magneticresonance, crystallography or photoaffinity labeling. See, for example,de Vos et al., Science 255:306-312, 1992; Smith et al., J. Mol. Biol.224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992.

[0096] In addition, libraries of KSE336 and fragments thereof can beused for screening and selection of KSE336 variants. For instance, alibrary of coding sequences can be generated by treating adouble-stranded DNA with a nuclease under conditions where the nickingoccurs, e.g., only once per molecule, denaturing the double-strandedDNA, renaturing it to for double-stranded DNA that can includesense/antisense pairs from different nicked products, removingsingle-stranded portions from reformed duplexes by treatment with S1nuclease, and ligating the resulting DNAs into an expression vecore. Bythis method, xpression libraries can be made comprising “mutagenized”KSE336. The entire coding sequence or parts thereof can be used.

[0097] Polynucleotide Expression, Polypeptides Produced Thereby, andSpecific-Binding Partners Thereto.

[0098] A polynucleotide according to the present invention can beexpressed in a variety of different systems, in vitro and in vivo,according to the desired purpose. For example, a polynucleotide can beinserted into an expression vector, introduced into a desired host, andcultured under conditions effective to achieve expression of apolypeptide coded for by the polynucleotide, to search for specificbinding partners. Effective conditions include any culture conditionswhich are suitable for achieving production of the polypeptide by thehost cell, including effective temperatures, pH, medium, additives tothe media in which the host cell is cultured (e.g., additives whichamplify or induce expression such as butyrate, or methotrexate if thecoding polynucleotide is adjacent to a dhfr gene), cycloheximide, celldensities, culture dishes, etc. A polynucleotide can be introduced intothe cell by any effective method including, e.g., naked DNA, calciumphosphate precipitation, electroporation, injection, DEAE-Dextranmediated transfection, fusion with liposomes, association with agentswhich enhance its uptake into cells, viral transfection. A cell intowhich a polynucleotide of the present invention has been introduced is atransformed host cell. The polynucleotide can be extrachromosomal orintegrated into a chromosome(s) of the host cell. It can be stable ortransient. An expression vector is selected for its compatibility withthe host cell. Host cells include, mammalian cells, e.g., COS, CV1, BHK,CHO, HeLa, LTK, NIH 3T3, CNS neural stem cells (e.g., U.S. Pat. No.6,103,530), IMR-32, A172 (ATCC CRL-1620), T98G (ATCC CRL-1690),CCF-STTGI (ATCC CRL-1718), DBTRG-05MG (ATCC CRL-2020), PFSK-1 (ATCCCRL-2060), SK-N-AS and other SK cell lines (ATCC CRL-2137), CHP-212(ATCC CRL-2273), RG2 (ATCC CRL-2433), HCN-2 (ATCC CRL-10742), U-87 MGand other U MG cell lines (ATCC HTB-14), D283 Med (ATCC HTB-185), PC12,Neuro-2a (ATCC CCL-131), insulinoma cell lines, INS-HI, MIN6N8, RIN-5AH,RIN-A12, RINm5F, capan-1, capan-2, MIA PaCa-2 (ATCC CRL-1420), PANC-1(ATCC CRL-1469), AsPC-1 (ATCC CRL-1682), SU-86.86 (ATCC CRL-1837),CFPAC-1 (ATCC CRL-1918), HPAF-II (ATCC CRL-1937), TGP61 (ATCC CRL-2135)and other TGP lines, SW 1990 (ATCC CRL-2172), Mpanc-96 (ATCC CRL-2380),MS1 VEGF (ATCC CRL-2460), Beta-TC-6 (ATCC CRL-11506), LTPA (ATCCCRL-2389), 266-6 (ATCC CRL-2151), MS1 (ATCC CRL-2779), SVR (ATCCCRL-2280), NIT-2 (ATCC CRL-2364), alphaTC1 Clone 9 (ATCC CRL-2350), ATCCCRL-1492, BxPC-3 (ATCC CRL-1687), HPAC (ATCC CRL-2119), U.S. Pat. Nos.6,110743, 5,928,942, 5,888,816, 5,888,705, and 5,723,333, etc., insectcells, such as Sf9 (S. frugipeda) and Drosophila, bacteria, such as E.coli, Streptococcus, bacillus, yeast, such as Sacharomyces, S.cerevisiae, fungal cells, plant cells, embryonic or adult stem cells(e.g., mammalian, such as mouse or human), pluripotent cells, etc.

[0099] Expression control sequences are similarly selected for hostcompatibility and a desired purpose, e.g., high copy number, highamounts, induction, amplification, controlled expression. Othersequences which can be employed include enhancers such as from SV40,CMV, RSV, inducible promoters, cell-type specific elements, or sequenceswhich allow selective or specific cell expression. Promoters that can beused to drive its expression, include, e.g., the endogenous promoter,MMTV, SV40, trp, lac, tac, or T7 promoters for bacterial hosts; or alphafactor, alcohol oxidase, or PGH promoters for yeast. RNA promoters canbe used to produced RNA transcripts, such as T7 or SP6. See, e.g.,Melton et al., Polynucleotide Res., 12(18):7035-7056, 1984; Dunn andStudier. J. Mol. Bio., 166:477-435, 1984; U.S. Pat. No. 5,891,636;Studier et al., Gene Expression Technology, Methods in Enzymology,85:60-89, 1987. In addition, as discussed above, translational signals(including in-frame insertions) can be included.

[0100] When a polynucleotide is expressed as a heterologous gene in atransfected cell line, the gene is introduced into a cell as describedabove, under effective conditions in which the gene is expressed. Theterm “heterologous” means that the gene has been introduced into thecell line by the “hand-of-man.” Introduction of a gene into a cell lineis discussed above. The transfected (or transformed) cell expressing thegene can be lysed or the cell line can be used intact.

[0101] For expression and other purposes, a polynucleotide can containcodons found in a naturally-occurring gene, transcript, or CDNA, forexample, e.g., as set forth in SEQ ID NO 1 and 2, or it can containdegenerate codons coding for the same amino acid sequences. Forinstance, it may be desirable to change the codons in the sequence tooptimize the sequence for expression in a desired host. See, e.g., U.S.Pat. Nos. 5,567,600 and 5,567,862.

[0102] A polypeptide according to the present invention can be recoveredfrom natural sources, transformed host cells (culture medium or cells)according to the usual methods, including, detergent extraction (e.g.,non-ionic detergent, Triton X-100, CHAPS, octylglucoside, Igepa1CA-630), ammonium sulfate or ethanol precipitation, acid extraction,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, hydroxyapatitechromatography, lectin chromatography, gel electrophoresis. Proteinrefolding steps can be used, as necessary, in completing theconfiguration of the mature protein. Finally, high performance liquidchromatography (HPLC) can be employed for purification steps. Anotherapproach is express the polypeptide recombinantly with an affinity tag(Flag epitope, HA epitope, myc epitope, 6xHis, maltose binding protein,chitinase, etc) and then purify by anti-tag antibody-conjugated affinitychromatography.

[0103] The present invention also relates to antibodies, and otherspecific-binding partners, which are specific for polypeptides encodedby polynucleotides of the present invention, e.g., KSE336. Antibodies,e.g., polyclonal, monoclonal, recombinant, chimeric, humanized,single-chain, Fab, and fragments thereof, can be prepared according toany desired method. See, also, screening recombinant immunoglobulinlibraries (e.g., Orlandi et al., Proc. Natl. Acad. Sci., 86:3833-3837,1989; Huse et al., Science, 256:1275-1281, 1989); in vitro stimulationof lymphocyte populations; Winter and Milstein, Nature, 349: 293-299,1991. The antibodies can be IgM, IgG, subtypes, IgG2a, IgG1, etc.Antibodies, and immune responses, can also be generated by administeringnaked DNA See, e.g., U.S. Pat. Nos. 5,703,055; 5,589,466; 5,580,859.Antibodies can be used from any source, including, goat, rabbit, mouse,chicken (e.g., IgY; see, Duan, WO/029444 for methods of makingantibodies in avian hosts, and harvesting the antibodies from the eggs).An antibody specific for a polypeptide means that the antibodyrecognizes a defined sequence of amino acids within or including thepolypeptide. Other specific binding partners include, e.g., aptamers andPNA. antibodies can be prepared against specific epitopes or domains ofKSE336, e.g., SEQ ID NO 4. The preparation of polyclonal antibodies iswell-known to those skilled in the art. See, for example, Green et al.,Production of Polyclonal Antisera, in IMMUNOCHEMICAL PROTOCOLS (Manson,ed.), pages 1-5 (Humana Press 1992); Coligan et al., Production ofPolyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in CURRENTPROTOCOLS IN IMMUNOLOGY, section 2.4.1 (1992). The preparation ofmonoclonal antibodies likewise is conventional. See, for example, Kohler& Milstein, Nature 256:495 (1975); Coligan et al., sections 2.5.1-2.6.7;and Harlow et al., ANTIBODIES: A LABORATORY MANUAL, page 726 (ColdSpring Harbor Pub. 1988).

[0104] Antibodies can also be humanized, e.g., where they are to be usedtherapeutically. Humanized monoclonal antibodies are produced bytransferring mouse complementarity determining regions from heavy andlight variable chains of the mouse immunoglobulin into a human variabledomain, and then substituting human residues in the framework regions ofthe murine counterparts. The use of antibody components derived fromhumanized monoclonal antibodies obviates potential problems associatedwith the immunogenicity of murine constant regions. General techniquesfor cloning murine immunoglobulin variable domains are described, forexample, by Orlandi et al., Proc. Nat'l Acad. Sci. USA 86:3833 (1989),which is hereby incorporated in its entirety by reference. Techniquesfor producing humanized monoclonal antibodies are described, forexample, in U.S. Pat. No. 6,054,297, Jones et al., Nature 321: 522(1986); Riechmann et al., Nature 332: 323 (1988); Verhoeyen et al.,Science 239: 1534 (1988); Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285 (1992); Sandhu, Crit. Rev. Biotech. 12: 437 (1992); and Singer etal., J. Immunol. 150: 2844 (1993).

[0105] Antibodies of the invention also may be derived from humanantibody fragments isolated from a combinatorial immunoglobulin library.See, for example, Barbas et al., METHODS: A COMPANION TO METHODS INENZYMOLOGY, VOL. 2, page 119 (1991); Winter et al., Ann. Rev. Immunol.12: 433 (1994). Cloning and expression vectors that are useful forproducing a human immunoglobulin phage library can be obtainedcommercially, for example, from STRATAGENE Cloning Systems (La Jolla,Calif.).

[0106] In addition, antibodies of the present invention may be derivedfrom a human monoclonal antibody. Such antibodies are obtained fromtransgenic mice that have been “engineered” to produce specific humanantibodies in response to antigenic challenge. In this technique,elements of the human heavy and light chain loci are introduced intostrains of mice derived from embryonic stem cell lines that containtargeted disruptions of the endogenous heavy and light chain loci. Thetransgenic mice can synthesize human antibodies specific for humanantigens and can be used to produce human antibody-secreting hybridomas.Methods for obtaining human antibodies from transgenic mice aredescribed, e.g., in Green et al., Nature Genet. 7:13 (1994); Lonberg etal., Nature 368:856 (1994); and Taylor et al., Int. Immunol. 6:579(1994).

[0107] Antibody fragments of the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ofnucleic acid encoding the fragment. Antibody fragments can be obtainedby pepsin or papain digestion of whole antibodies by conventionalmethods. For example, antibody fragments can be produced by enzymaticcleavage of antibodies with pepsin to provide a 5S fragment denotedF(ab′).sub.2. This fragment can be further cleaved using a thiolreducing agent, and optionally a blocking group for the sulfhydrylgroups resulting from cleavage of disulfide linkages, to produce 3.5SFab′ monovalent fragments. Alternatively, an enzymatic cleavage usingpepsin produces two monovalent Fab′ fragments and an Fc fragmentdirectly. These methods are described, for example, by Goldenberg, U.S.Pat. Nos. 4,036,945 and 4,331,647, and references contained therein.These patents are hereby incorporated in their entireties by reference.See also Nisoiihoff et al., Arch. Biochem. Biophys. 89:230 (1960);Porter, Biochem. J. 73:119 (1959); Edelman et al, METHODS IN ENZYMOLOGY,VOL. 1, page 422 (Academic Press 1967); and Coligan et al. at sections2.8.1-2.8.10 and 2.10.1-2.10.4.

[0108] Other methods of cleaving antibodies, such as separation of heavychains to form monovalent light-heavy chain fragments, further cleavageof fragments, or other enzymatic, chemical, or genetic techniques canalso be used. For example, Fv fragments comprise an association ofV.sub.H and V.sub.L chains. This association may be noncovalent, asdescribed in Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659 (1972).Alternatively, the variable chains can be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde. See,e.g., Sandhu, supra. Preferably, the Fv fragments comprise V.sub.H andV.sub.L chains connected by a peptide linker. These single-chain antigenbinding proteins (sFv) are prepared by constructing a structural genecomprising nucleic acid sequences encoding the V.sub.H and V.sub.Ldomains connected by an oligonucleotide. The structural gene is insertedinto an expression vector, which is subsequently introduced into a hostcell such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by Whitlow etal., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 97(1991); Bird et al.,Science 242:423-426 (1988); Ladneret al., U.S. Pat.No. 4,946,778; Pack et al., Bio/Technology 11: 1271-77 (1993); andSandhu, supra.

[0109] Another form of an antibody fragment is a peptide coding for asingle complementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick et al.,METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 106 (1991).

[0110] The term “antibody” as used herein includes intact molecules aswell as fragments thereof, such as Fab, F(ab′)2, and Fv which arecapable of binding to an epitopic determinant present in Binlpolypeptide. Such antibody fragments retain some ability to selectivelybind with its antigen or receptor. The term “epitope” refers to anantigenic determinant on an antigen to which the paratope of an antibodybinds. Epitopic determinants usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Antibodies can be preparedagainst specific epitopes or polypeptide domains.

[0111] Antibodies which bind to KSE336 polypeptides of the presentinvention can be prepared using an intact polypeptide or fragmentscontaining small peptides of interest as the immunizing antigen. Forexample, it may be desirable to produce antibodies that specificallybind to the N- or C-terminal domains of KSE336. The polypeptide orpeptide used to immunize an animal which is derived from translated cDNAor chemically synthesized which can be conjugated to a carrier protein,if desired. Such commonly used carriers which are chemically coupled tothe immunizing peptide include keyhole limpet hemocyanin (KLH),thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.

[0112] Polyclonal or monoclonal antibodies can be further purified, forexample, by binding to and elution from a matrix to which thepolypeptide or a peptide to which the antibodies were raised is bound.Those of skill in the art will know of various techniques common in theimmunology arts for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies (See for example, Coligan,et al., Unit 9, Current Protocols in Immunology, Wiley Interscience,1994, incorporated by reference).

[0113] Anti-idiotype technology can also be used to produce inventionmonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody.

[0114] Methods of Detecting Polypeptides

[0115] Polypeptides coded for by KSE336 of the present invention can bedetected, visualized, determined, quantitated, etc. according to anyeffective method. useful methods include, e.g., but are not limited to,immunoassays, RIA (radioimmunassay), ELISA, (enzyme-linked-immunosorbentassay), immunoflourescence, flow cytometry, histology, electronmicroscopy, light microscopy, in situ assays, immunoprecipitation,Western blot, etc.

[0116] Immunoassays may be carried in liquid or on biological support.For instance, a sample (e.g., blood, stool, urine, cells, tissue, bodyfluids, etc.) can be brought in contact with and immobilized onto asolid phase support or carrier such as nitrocellulose, or other solidsupport that is capable of immobilizing cells, cell particles or solubleproteins. The support may then be washed with suitable buffers followedby treatment with the detectably labeled KSE336 specific antibody. Thesolid phase support can then be washed with a buffer a second time toremove unbound antibody. The amount of bound label on solid support maythen be detected by conventional means.

[0117] A “solid phase support or carrier” includes any support capableof binding an antigen, antibody, or other specific binding partner.Supports or carriers include glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, and magnetite. A support material can have anystructural or physical configuration. Thus, the support configurationmay be spherical, as in a bead, or cylindrical, as in the inside surfaceof a test tube, or the external surface of a rod. Alternatively, thesurface may be flat such as a sheet, test strip, etc. Preferred supportsinclude polystyrene beads

[0118] One of the many ways in which gene peptide-specific antibody canbe detectably labeled is by linking it to an enzyme and using it in anenzyme immunoassay (EIA). See, e.g., Voller, A., “The Enzyme LinkedImmunosorbent Assay (ELISA),” 1978, Diagnostic Horizons 2, 1-7,Microbiological Associates Quarterly Publication, Walkersville, Md.);Voller, A. et al., 1978, J. Clin. Pathol. 31, 507-520; Butler, J. E.,1981, Meth. Enzymol. 73, 482-523; Maggio, E. (ed.), 1980, EnzymeImmunoassay, CRC Press, Boca Raton, Fla. The enzyme which is bound tothe antibody will react with an appropriate substrate, preferably achromogenic substrate, in such a manner as to produce a chemical moietythat can be detected, for example, by spectrophotometric, fluorimetricor by visual means. Enzymes that can be used to detectably label theantibody include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, .alpha.-glycerophosphate, dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, .beta.-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods that employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

[0119] Detection may also be accomplished using any of a variety ofother immunoassays. For example, by radioactively labeling theantibodies or antibody fragments, it is possible to detect KSE336peptides through the use of a radioimmunoassay (RIA). See, e.g.,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986. Theradioactive isotope can be detected by such means as the use of a gammacounter or a scintillation counter or by autoradiography.

[0120] It is also possible to label the antibody with a fluorescentcompound. When the fluorescently labeled antibody is exposed to light ofthe proper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. Theantibody can also be detectably labeled using fluorescence emittingmetals such as those in the lanthanide series. These metals can beattached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

[0121] The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples of usefulchemiluminescent labeling compounds are luminol, isoluminol, theromaticacridinium ester, imidazole, acridinium salt and oxalate ester.

[0122] Likewise, a bioluminescent compound may be used to label theantibody of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase and aequorin.

[0123] Diagnostic

[0124] The present invention also relates to methods and compositionsfor diagnosing a brain or pancreas disorder, or determining whether asubject is susceptible to such a disorder, using polynucleotides,polypeptides, and specific-binding partners of the present invention todetect, assess, determine, etc., KSE336. In such methods, the gene canserve as a marker for the disorder, e.g., where the gene, when mutant,is a direct cause of the disorder; where the gene is affected by anothergene(s) which is directly responsible for the disorder, e.g., when thegene is part of the same signaling pathway as the directly responsiblegene; and, where the gene is chromosomally linked to the gene(s)directly responsible for the disorder, and segregates with it. Manyother situations are possible. To detect, assess, determine, etc., aprobe specific for the gene can be employed as described above andbelow. Any method of detecting and/or assessing the gene can be used,including detecting expression of the gene using polynucleotides,antibodies, or other specific-binding partners.

[0125] The present invention relates to methods of diagnosing a disorderassociated with a disorder of KSE336, or determining whether a subjectis susceptible to such a disorder, comprising, e.g., assessing theexpression of KSE336 in a tissue sample comprising tissue or cellssuspected of having the disorder (e.g., where the sample comprises brainand pancreas). The phrase “diagnosing” indicates that it is determinedwhether the sample has the disorder. A “disorder” means, e.g., anyabnormal condition as in a disease or malady. “Determining a subject'ssusceptibility to a disease or disorder” indicates that the subject isassessed for whether s/he is predisposed to get such a disease ordisorder, where the predisposition is indicated by abnormal expressionof the gene (e.g., gene mutation, gene expression pattern is not normal,etc.). Predisposition or susceptibility to a disease may result when asuch disease is influenced by epigenetic, environmental, etc., factors.

[0126] By the phrase “assessing expression of KSE336,” it is meant thatthe functional status of the gene is evaluated. This includes, but isnot limited to, measuring expression levels of said gene, determiningthe genomic structure of said gene, determining the mRNA structure oftranscripts from said gene, or measuring the expression levels ofpolypeptide coded for by said gene. Thus, the term “assessingexpression” includes evaluating the all aspects of the transcriptionaland translational machinery of the gene. For instance, if a promoterdefect causes, or is suspected of causing, the disorder, then a samplecan be evaluated (i.e., “assessed”) by looking (e.g., sequencing orrestriction mapping) at the promoter sequence in the gene, by detectingtranscription products (e.g., RNA), by detecting translation product(e.g., polypeptide). Any measure of whether the gene is functional canbe used, including, polypeptide, polynucleotide, and functional assaysfor the gene's biological activity.

[0127] In making the assessment, it can be useful to compare the resultsto a normal gene, e.g., a gene which is not associated with thedisorder. The nature of the comparison can be determined routinely,depending upon how the assessing is accomplished. If, for example, themRNA levels of a sample is detected, then the mRNA levels of a normalcan serve as a comparison, or a gene which is known not to be affectedby the disorder. Methods of detecting mRNA are well known, and discussedabove, e.g., but not limited to, Northern blot analysis, polymerasechain reaction (PCR), reverse transcriptase PCR, RACE PCR, etc.Similarly, if polypeptide production is used to evaluate the gene, thenthe polypeptide in a normal tissue sample can be used as a comparison,or, polypeptide from a different gene whose expression is known not tobe affected by the disorder. These are only examples of how such amethod could be carried out.

[0128] Assessing the effects of therapeutic and preventativeinterventions (e.g., administration of a drug, chemotherapy, radiation,etc.) on brain and pancreas disorders is a major effort in drugdiscovery, clinical medicine, and pharmacogenomics. The evaluation oftherapeutic and preventative measures, whether experimental or alreadyin clinical use, has broad applicability, e.g., in clinical trials, formonitoring the status of a patient, for analyzing and assessing animalmodels, and in any scenario involving cancer treatment and prevention.Analyzing the expression profiles of polynucleotides of the presentinvention can be utilized as a parameter by which interventions arejudged and measured. Treatment of a disorder can change the expressionprofile in some manner which is prognostic or indicative of the drug'seffect on it. Changes in the profile can indicate, e.g., drug toxicity,return to a normal level, etc. Accordingly, the present invention alsorelates to methods of monitoring or assessing a therapeutic orpreventative measure (e.g., chemotherapy, radiation, anti-neoplasticdrugs, antibodies, etc.) in a subject having a brain and pancreasdisorder, or, susceptible to such a disorder, comprising, e.g.,detecting the expression levels of KSE336. A subject can be a cell-basedassay system, non-human animal model, human patient, etc. Detecting canbe accomplished as described for the methods above and below. By“therapeutic or preventative intervention,” it is meant, e.g., a drugadministered to a patient, surgery, radiation, chemotherapy, and othermeasures taken to prevent, treat, or diagnose a disorder.

[0129] Expression can be assessed in any sample comprising any tissue orcell type, body fluid, etc., as discussed for other methods of thepresent invention, including cells from brain and pancreas can be used,or cells derived from brain and pancreas. By the phrase “cells derivedfrom brain and pancreas,” it is meant that the derived cells originatefrom brain and pancreas, e.g., when metastasis from a primary tumor sitehas occurred, when a progenitor-type or pluripotent cell gives rise toother cells, etc.

[0130] Identifying Agent Methods

[0131] The present invention also relates to methods of identifyingagents that modulate the expression of KSE336 expressed in brain andpancreas cells, comprising, in any effective order, one or more of thefollowing steps, e.g., contacting a cell population with a test agentunder conditions effective for said test agent to modulate theexpression of KSE336 in said cell population, and determining whethersaid test agent modulates said KSE336. An agent can modulate expressionof KSE336 at any level, including transcription, translation, and/orperdurance of the nucleic acid or polypeptide (e.g., degradation,stability, etc.) product in the cell.

[0132] Contacting the cell population with the test agent can beaccomplished by any suitable method and/or means that places the agentin a position to functionally control expression of the KSE336 presentin cells within the population. Functional control indicates that theagent can exert its physiological effect on the cell through whatevermechanism it works. The choice of the method and/or means can dependupon the nature of the agent and the condition and type of the cellpopulation (such as, in vivo, in vitro, organ explants, etc.). Forinstance, if the cell population is an in vitro cell culture, the agentcan be contacted with the cells by adding it directly into the culturemedium. If the agent cannot dissolve readily in an aqueous medium, itcan be incorporated into liposomes, or another lipophilic carrier, andthen administered to the cell culture. Contact can also be facilitatedby incorporation of agent with carriers and delivery molecules andcomplexes, by injection, by infusion, etc.

[0133] After the agent has been administered in such a way that it cangain access to the cells, it can be determined whether the test agentmodulates KSE336 expression. Modulation can be of any type, quality, orquantity, e.g., increase, facilitate, enhance, up-regulate, stimulate,activate, amplify, augment, induce, decrease, down-regulate, diminish,lessen, reduce, etc. The modulatory quantity can also encompass anyvalue, e.g., 1%, 5%, 10%, 50%, 75%, 1-fold, 2-fold, 5-fold, 10-fold,100-fold, etc. To modulate KSE336 expression means, e.g., that the testagent has an effect on its expression, e.g., to effect the amount oftranscription, to effect RNA splicing, to effect translation of the RNAinto polypeptide, to effect RNA or polypeptide stability, to effectpolyadenylation or other processing of the RNA, to effectpost-transcriptional or post-translational processing, etc.

[0134] A test agent can be of any molecular composition, e.g., chemicalcompounds, biomolecules, such as polypeptides, lipids, nucleic acids(e.g., antisense to a polynucleotide sequence selected from SEQ ID NO 1and 2), carbohydrates, antibodies, ribozymes, double-stranded RNA, etc.For example, if a gene to be modulated is a cell-surface molecule, atest agent can be an antibody that specifically recognizes it and leadsto some effect on its expression. An antibody can cause the polypeptideto be internalized, leading to its down regulation on the surface of thecell. Such an effect does not have to be permanent, but can require thepresence of the antibody to continue the down-regulatory effect.Antisense KSE336 can also be used as test agents to modulate geneexpression.

[0135] Markers

[0136] The polynucleotides of the present invention can be used withother markers, especially brain and pancreas markers, to identity,detect, stage, diagnosis, determine, prognosticate, treat, etc., tissue,diseases and conditions, etc, of the brain and pancreas. Markers can bepolynucleotides, polypeptides, antibodies, ligands, specific bindingpartners, etc. The targets for such markers include, but are not limitedgenes and polypeptides that are selective for cell types present in thebrain and pancreas. Specific targets include, The targets for suchmarkers include, but are not limited, presenilins, genes andpolypeptides in the pathways for neurotransmitter synthesis, receptor,metabolism, etc., (e.g., serotonin, MAO, dopamine, norephinephrine,nitric oxide, etc.), apolipoprotein A, APP, neuron-specific enolase(NSE), glial fibrillary acidic protein (GFAP), S100, GAP-43,neuron-specific beta-III tubulin, Stac (neuron-specific protein with anSH3 domain, e.g., Genomics, 47:140-2, 1998), myelin basic protein, etc.vimentin, lannotti et al., Genomics, 46:520-524, 1997), ZG-46p (Chen etal., Eur. J. Cell. Biol., 3:205-214, 1997), calretinin, islet amyloidpancreatic polypeptide, SLC26A6 (e.g., on apical surface of pancreaticductal cells), reg/PSP mutligene family (e.g., Unno et al. J. Biol.Chem., 268:15974-82, 1993), pancreatitis-associated proteins (e.g.,Dusetti et al., Genomics, 19:108-114, 1994), PANC1A and PANC1B (U.S.Pat. No. 5,840,870), antibodies (e.g., U.S. Pat. No. 5,888,813 and5,622,837), INGAP (U.S. Pat. No. 5,840,531), insulin, glucagons, etc.

[0137] Therapeutics

[0138] Selective polynucleotides, polypeptides, and specific-bindingpartners thereto, can be utilized in therapeutic applications,especially to treat diseases and conditions of brain and pancreas.Useful methods include, but are not limited to, immunotherapy (e.g.,using specific-binding partners to polypeptides), vaccination (e.g.,using a selective polypeptide or a naked DNA encoding such polypeptide),protein or polypeptide replacement therapy, gene therapy (e.g.,germ-line correction, antisense), etc.

[0139] Various immunotherapeutic approaches can be used. For instance,unlabeled antibody that specifically recognizes a tissue-specificantigen can be used to stimulate the body to destroy or attack thecancer, to cause down-regulation, to produce complement-mediated lysis,to inhibit cell growth, etc., of target cells which display the antigen,e.g., analogously to how c-erbB-2 antibodies are used to treat breastcancer. In addition, antibody can be labeled or conjugated to enhanceits deleterious effect, e.g., with radionuclides and other energyemitting entitities, toxins, such as ricin, exotoxin A (ETA), anddiphtheria, cytotoxic or cytostatic agents, immunomodulators,chemotherapeutic agents, etc. See, e.g., U.S. Pat. No. 6,107,090.

[0140] An antibody or other specific-binding partner can be conjugatedto a second molecule, such as a cytotoxic agent, and used for targetingthe second molecule to a tissue-antigen positive cell (Vitetta, E. S. etal., 1993, Immunotoxin therapy, in DeVita, Jr., V. T. et al., eds,Cancer: Principles and Practice of Oncology, 4th ed., J. B. LippincottCo., Philadelphia, 2624-2636). Examples of cytotoxic agents include, butare not limited to, antimetabolites, alkylating agents, anthracyclines,antibiotics, anti-mitotic agents, radioisotopes and chemotherapeuticagents. Further examples of cytotoxic agents include, but are notlimited to ricin, doxorubicin, daunorubicin, taxol, ethidium bromide,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine,dihydroxy anthracin dione, actinomycin D, 1-dehydrotestosterone,diptheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, elongationfactor-2 and glucocorticoid. Techniques for conjugating therapeuticagents to antibodies are well.

[0141] In addition to immunotherapy, polynucleotides and polypeptidescan be used as targets for non-immunotherapeutic applications, e.g.,using compounds which interfere with function, expression (e.g.,antisense as a therapeutic agent), assembly, etc. RNA interference canbe used in vitro and in vivo to silence KSE336 when its expressioncontributes to a disease (but also for other purposes, e.g., to identifythe gene's function to change a developmental pathway of a cell, etc.).See, e.g., Sharp and Zamore, Science, 287:2431-2433, 2001; Grishok etal., Science, 287:2494, 2001.

[0142] Delivery of therapeutic agents can be achieved according to anyeffective method, including, liposomes, viruses, plasmid vectors,bacterial delivery systems, orally, systemically, etc.

[0143] In addition to therapeutics, per se, the present invention alsorelates to methods of treating a disease of the brain, pancreas, orprogenitor tissue thereof showing altered expression of KSE336,comprising, e.g., administering to a subject in need thereof atherapeutic agent which is effective for regulating expression of saidKSE336 and/or which is effective in treating said disease. The term“treating” is used conventionally, e.g., the management or care of asubject for the purpose of combating, alleviating, reducing, relieving,improving the condition of, etc., of a disease or disorder. Variousdisease can be treated, including, but not limited to, astrocytoma,meningioma, pancreatic adenocarcinoma, insulin-dependent diabetesmellitus 2 (IDDM2), helicoid peripapillary chorioretinal degeneration(also known as atrophia areata), Beckwith-Wiedemann syndrome, andcongenital hyperinsulinism.

[0144] By the phrase “altered expression,” it is meant that the diseaseis associated with a mutation in the gene, or any modification to thegene (or corresponding product) which affects its normal function. Thus,expression of KSE336 refers to, e.g., transcription, translation,splicing, stability of the mRNA or protein product, activity of the geneproduct, differential expression, etc.

[0145] Any agent which “treats” the disease can be used. Such an agentcan be one which regulates the expression of the KSE336. Expressionrefers to the same acts already mentioned, e.g. transcription,translation, splicing, stability of the mRNA or protein product,activity of the gene product, differential expression, etc. Forinstance, if the condition was a result of a complete deficiency of thegene product, administration of gene product to a patient would be saidto treat the disease and regulate the gene's expression. Many otherpossible situations are possible, e.g., where the gene is aberrantlyexpressed, and the therapeutic agent regulates the aberrant expressionby restoring its normal expression pattern.

[0146] Antisense

[0147] Antisense polynucleotide (e.g., RNA) can also be prepared from apolynucleotide according to the present invention, preferably ananti-sense to a sequence of SEQ ID NO 1 and 2. Antisense polynucleotidecan be used in various ways, such as to regulate or modulate expressionof the polypeptides they encode, e.g., inhibit their expression, for insitu hybridization, for therapeutic purposes, for making targetedmutations (in vivo, triplex, etc.) etc. For guidance on administeringand designing anti-sense, see, e.g., U.S. Pat. Nos. 6,200,960,6,200,807, 6,197,584, 6,190,869, 6,190,661, 6,187,587, 6,168,950,6,153,595, 6,150,162, 6,133,246, 6,117,847, 6,096,722, 6,087,343,6,040,296, 6,005,095, 5,998,383, 5,994,230, 5,891,725, 5,885,970, and5,840,708. An antisense polynucleotides can be operably linked to anexpression control sequence. A total length of about 35 bp can be usedin cell culture with cationic liposomes to facilitate cellular uptake,but for in vivo use, preferably shorter oligonucleotides areadministered, e.g. 25 nucleotides.

[0148] Antisense polynucleotides can comprise modified,nonnaturally-occurring nucleotides and linkages between the nucleotides(e.g., modification of the phosphate-sugar backbone; methyl phosphonate,phosphorothioate, or phosphorodithioate linkages; and 2′-O-methyl ribosesugar units), e.g., to enhance in vivo or in vitro stability, to confernuclease resistance, to modulate uptake, to modulate cellulardistribution and compartmentalization, etc. Any effective nucleotide ormodification can be used, including those already mentioned, as known inthe art, etc., e.g., disclosed in U.S. Pat. Nos. 6,133,438; 6,127,533;6,124,445; 6,121,437; 5,218,103 (e.g., nucleoside thiophosphoramidites);4,973,679; Sproat et al., “2′-O-Methyloligoribonucleotides: synthesisand applications,” Oligonucleotides and Analogs A Practical Approach,Eckstein (ed.), IRL Press, Oxford, 1991, 49-86; Iribarren et al.,“2′O-Alkyl Oligoribonucleotides as Antisense Probes,” Proc. Natl. Acad.Sci. USA, 1990, 87, 7747-7751; Cotton et al., “2′-O-methyl, 2′-O-ethyloligoribonucleotides and phosphorothioate oligodeoxyribonucleotides asinhibitors of the in vitro U7 snRNP-dependent mRNA processing event,”Nucl. Acids Res., 1991, 19, 2629-2635.

[0149] Arrays

[0150] The present invention also relates to an ordered array ofpolynucleotide probes and specific-binding partners (e.g., antibodies)for detecting the expression of KSE336 in a sample, comprising, one ormore polynucleotide probes or specific binding partners associated witha solid support, wherein each probe is specific for KSE336, and theprobes comprise a nucleotide sequence of SEQ ID NO 1 and 2 which isspecific for said gene, a nucleotide sequence having sequence identityto SEQ ID NO 1 and 2 which is specific for said gene or polynucleotide,or complements thereto, or a specific-binding partner which is specificfor KSE336.

[0151] The phrase “ordered array” indicates that the probes are arrangedin an identifiable or position-addressable pattern, e.g., such as thearrays disclosed in U.S. Pat. Nos. 6,156,501, 6,077,673, 6,054,270,5,723,320, 5,700,637, WO09919711, WO00023803. The probes are associatedwith the solid support in any effective way. For instance, the probescan be bound to the solid support, either by polymerizing the probes onthe substrate, or by attaching a probe to the substrate. Association canbe, covalent, electrostatic, noncovalent, hydrophobic, hydrophilic,noncovalent, coordination, adsorbed, absorbed, polar, etc. When fibersor hollow filaments are utilized for the array, the probes can fill thehollow orifice, be absorbed into the solid filament, be attached to thesurface of the orifice, etc. Probes can be of any effective size,sequence identity, composition, etc., as already discussed.

[0152] Ordered arrays can further comprise polynucleotide probes orspecific-binding partners which are specific for other genes, includinggenes specific for brain and pancreas or disorders associated with brainand pancreas.

[0153] Transgenic Animals

[0154] The present invention also relates to transgenic animalscomprising KSE336 genes. Such genes, as discussed in more detail below,include, but are not limited to, functionally-disrupted genes, mutatedgenes, ectopically or selectively-expressed genes, inducible orregulatable genes, etc. These transgenic animals can be producedaccording to any suitable technique or method, including homologousrecombination, mutagenesis (e.g., ENU, Rathkolb et al., Exp. Physiol.,85(6):635-644, 2000), and the tetracycline-regulated gene expressionsystem (e.g., U.S. Pat. No. 6,242,667). The term “gene” as used hereinincludes any part of a gene, i.e., regulatory sequences, promoters,enhancers, exons, introns, coding sequences, etc. The KSE336 nucleicacid present in the construct or transgene can be naturally-occurringwild-type, polymorphic, or mutated.

[0155] Along these lines, polynucleotides of the present invention canbe used to create transgenic animals, e.g. a non-human animal,comprising at least one cell whose genome comprises a functionaldisruption of KSE336. By the phrases “functional disruption” or“functionally disrupted,” it is meant that the gene does not express abiologically-active product. It can be substantially deficient in atleast one functional activity coded for by the gene. Expression of apolypeptide can be substantially absent, i.e., essentially undetectableamounts are made. However, polypeptide can also be made, but which isdeficient in activity, e.g., where only an amino-terminal portion of thegene product is produced.

[0156] The transgenic animal can comprise one or more cells. Whensubstantially all its cells contain the engineered gene, it can bereferred to as a transgenic animal “whose genome comprises” theengineered gene. This indicates that the endogenous gene loci of theanimal has been modified and substantially all cells contain suchmodification.

[0157] Functional disruption of the gene can be accomplished in anyeffective way, including, e.g., introduction of a stop codon into anypart of the coding sequence such that the resulting polypeptide isbiologically inactive (e.g., because it lacks a catalytic domain, aligand binding domain, etc.), introduction of a mutation into a promoteror other regulatory sequence that is effective to turn it off, or reducetranscription of the gene, insertion of an exogenous sequence into thegene which inactivates it (e.g., which disrupts the production of abiologically-active polypeptide or which disrupts the promoter or othertranscriptional machinery), deletion of sequences from the KSE336 gene,etc. Examples of transgenic animals having functionally disrupted genesare well known, e.g., as described in U.S. Pat. Nos. 6,239,326,6,225,525, 6,207,878, 6,194,633, 6,187,992, 6,180,849, 6,177,610,6,100,445, 6,087,555, 6,080,910, 6,069,297, 6,060,642, 6,028,244,6,013,858, 5,981,830, 5,866,760, 5,859,314, 5,850,004, 5,817,912,5,789,654, 5,777,195, and 5,569,824. A transgenic animal which comprisesthe functional disruption can also be referred to as a “knock-out”animal, since the biological activity of its KSE336 genes has been“knocked-out.” Knock-outs can be homozygous or heterozygous.

[0158] For creating functional disrupted genes, and other genemutations, homologous recombination technology is of special interestsince it allows specific regions of the genome to be targeted. Usinghomologous recombination methods, genes can be specifically-inactivated,specific mutations can be introduced, and exogenous sequences can beintroduced at specific sites. These methods are well known in the art,e.g., as described in the patents above. See, also, Robertson, Biol.Reproduc., 44(2):238-245, 1991. Generally, the genetic engineering isperformed in an embryonic stem (ES) cell, or other pluripotent cell line(e.g., adult stem cells, EG cells), and that genetically-modified cell(or nucleus) is used to create a whole organism. Nuclear transfer can beused in combination with homologous recombination technologies.

[0159] For example, the KSE336 locus can be disrupted in mouse ES cellsusing a positive-negative selection method (e.g., Mansour et al.,Nature, 336:348-352, 1988). In this method, a targeting vector can beconstructed which comprises a part of the gene to be targeted. Aselectable marker, such as neomycin resistance genes, can be insertedinto a KSE336 exon present in the targeting vector, disrupting it. Whenthe vector recombines with the ES cell genome, it disrupts the functionof the gene. The presence in the cell of the vector can be determined byexpression of neomycin resistance. See, e.g., U.S. Pat. No. 6,239,326.Cells having at least one functionally disrupted gene can be used tomake chimeric and germline animals, e.g., animals having somatic and/orgerm cells comprising the engineered gene. Homozygous knock-out animalscan be obtained from breeding heterozygous knock-out animals. See, e.g.,U.S. Pat. No. 6,225,525.

[0160] A transgenic animal, or animal cell, lacking one or morefunctional KSE336 genes can be useful in a variety of applications,including, as an animal model for brain and pancreas diseases, for drugscreening assays (e.g., for kinases other than KSE336; by making a celldeficient in KSE336, the contribution of other kinases can bespecifically examined), as a source of tissues deficient in KSE336activity, and any of the utilities mentioned in any issued U.S. Patenton transgenic animals, including, U.S. Pat. Nos. 6,239,326, 6,225,525,6,207,878, 6,194,633, 6,187,992, 6,180,849, 6,177,610, 6,100,445,6,087,555, 6,080,910, 6,069,297, 6,060,642, 6,028,244, 6,013,858,5,981,830, 5,866,760, 5,859,314, 5,850,004, 5,817,912, 5,789,654,5,777,195, and 5,569,824. For instance, KSE336 deficient animal cellscan be utilized to study kinase activities. Pancreas and brain cellsdisplay a variety of enzyme activities which are responsive toextracellular and intracellular signals. By knocking-out protein kinaseactivity, e.g., one at a time, the physiological pathways using kinaseactivity can be dissected out and identified.

[0161] The present invention also relates to non-human, transgenicanimal whose genome comprises recombinant KSE336 nucleic acidoperatively linked to an expression control sequence effective toexpress said coding sequence, e.g., in pancreas and brain. Such atransgenic animal can also be referred to as a “knock-in” animal sincean exogenous gene has been introduced, stably, into its genome.

[0162] A recombinant KSE336 nucleic acid refers to a gene which has beenintroduced into a target host cell and optionally modified, such ascells derived from animals, plants, bacteria, yeast, etc. A recombinantKSE336 includes completely synthetic nucleic acid sequences,semi-synthetic nucleic acid sequences, sequences derived from naturalsources, and chimeras thereof. “Operable linkage” has the meaning usedthrough the specification, i.e., placed in a functional relationshipwith another nucleic acid. When a gene is operably linked to anexpression control sequence, as explained above, it indicates that thegene (e.g., coding sequence) is joined to the expression controlsequence (e.g., promoter) in such a way that facilitates transcriptionand translation of the coding sequence. As described above, the phrase“genome” indicates that the genome of the cell has been modified. Inthis case, the recombinant KSE336 has been stably integrated into thegenome of the animal. The KSE336 nucleic acid in operable linkage withthe expression control sequence can also be referred to as a constructor transgene.

[0163] Any expression control sequence can be used depending on thepurpose. For instance, if selective expression is desired, thenexpression control sequences which limit its expression can be selected.These include, e.g., tissue or cell-specific promoters, introns,enhancers, etc. For various methods of cell and tissue-specificexpression, see, e.g., U.S. Pat. Nos. 6,215,040, 6,210,736, and6,153,427. These also include the endogenous promoter, i.e., the codingsequence can be operably linked to its own promoter. Inducible andregulatable promoters can also be utilized.

[0164] The present invention also relates to a transgenic animal whichcontains a functionally disrupted and a transgene stably integrated intothe animals genome. Such an animal can be constructed using combinationsany of the above- and below-mentioned methods. Such animals have any ofthe aforementioned uses, including permitting the knock-out of thenormal gene and its replacement with a mutated gene. Such a transgenecan be integrated at the endogenous gene locus so that the functionaldisruption and “knock-in” are carried out in the same step.

[0165] In addition to the methods mentioned above, transgenic animalscan be prepared according to known methods, including, e.g., bypronuclear injection of recombinant genes into pronuclei of 1-cellembryos, incorporating an artificial yeast chromosome into embryonicstem cells, gene targeting methods, embryonic stem cell methodology,cloning methods, nuclear transfer methods. See, also, e.g., U.S. Pat.Nos. 4,736,866; 4,873,191; 4,873,316; 5,082,779; 5,304,489; 5,174,986;5,175,384; 5,175,385; 5,221,778; Gordon et al., Proc. Natl. Acad. Sci.,77:7380-7384, 1980; Palmiter et al., Cell, 41:343-345, 1985; Palmiter etal., Ann. Rev. Genet., 20:465-499, 1986; Askew et al., Mol. Cell. Bio.,13:4115-4124, 1993; Games et al. Nature, 373:523-527, 1995; Valanciusand Smithies, Mol. Cell. Bio., 11:1402-1408, 1991; Stacey et al., Mol.Cell. Bio., 14:1009-1016, 1994; Hasty et al., Nature, 350:243-246, 1995;Rubinstein et al., Nucl. Acid Res., 21:2613-2617,1993; Cibelli et al.,Science, 280:1256-1258, 1998. For guidance on recombinase excisionsystems, see, e.g., U.S. Pat. Nos. 5,626,159, 5,527,695, and 5,434,066.See also, Orban, P.C., et al., “Tissue- and Site-Specific DNARecombination in Transgenic Mice,” Proc. Natl. Acad. Sci. USA,89:6861-6865 (1992); O'Gorman, S., et al., “Recombinase-Mediated GeneActivation and Site-Specific Integration in Mammalian Cells,” Science,251:1351-1355 (1991); Sauer, B., et al., “Cre-stimulated recombinationat loxP-Containing DNA sequences placed into the mammalian genome,”Polynucleotides Research, 17(1):147-161 (1989); Gagneten, S. et al.(1997) Nucl. Acids Res. 25:3326-3331; Xiao and Weaver (1997) Nucl. AcidsRes. 25:2985-2991; Agah, R. et al. (1997) J. Clin. Invest. 100: 169-179;Barlow, C. et al. (1997) Nucl. Acids Res. 25:2543-2545; Araki, K. et al.(1997) Nucl. Acids Res. 25:868-872; Mortensen, R. N. et al. (1992) Mol.Cell. Biol. 12:2391-2395 (G418 escalation method); Lakhlani, P. P. etal. (1997) Proc. Natl. Acad. Sci. USA 94:9950-9955 (“hit and run”);Westphal and Leder (1997) Curr. Biol. 7:530-533 (transposon-generated“knock-out” and “knock-in”); Templeton, N. S. et al. (1997) Gene Ther.4:700-709 (methods for efficient gene targeting, allowing for a highfrequency of homologous recombination events, e.g., without selectablemarkers); PCT International Publication WO 93/22443(functionally-disrupted).

[0166] A polynucleotide according to the present invention can beintroduced into any non-human animal, including a non-human mammal,mouse (Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1986), pig(Hammer et al., Nature, 315:343-345, 1985), sheep (Hammer et al.,Nature, 315:343-345, 1985), cattle, rat, or primate. See also, e.g.,Church, 1987, Trends in Biotech. 5:13-19; Clark et al., Trends inBiotech. 5:20-24, 1987); and DePamphilis et al., BioTechniques,6:662-680, 1988. Transgenic animals can be produced by the methodsdescribed in U.S. Pat. No. 5,994,618, and utilized for any of theutilities described therein.

[0167] The present invention relates to, e.g.,

[0168] a non-human, transgenic mammal whose genome comprises afunctional disruption of KSE336, optionally whose genome furthercomprises KSE336 operatively linked to an expression control sequenceeffective to express said gene in brain and pancreas cells, cellsderived from brain and pancreas, or brain and pancreas progenitor cells.and optionally, the expression control sequence is an induciblepromoter;

[0169] a mammalian cell whose genome comprises a functional disruptionof KSE336, and optionally, where the cell is a brain and pancreas, cellderived from brain and pancreas, or a brain and pancreas progenitorcell;

[0170] a non-human, transgenic mammal whose genome comprises arecombinant KSE336 nucleic acid operatively linked to an expressioncontrol sequence effective to express said gene in brain and pancreas,cells derived from brain and pancreas, or brain and pancreas progenitorcells, optionally, where the expression control sequence is an induciblepromoter, and optionally, whose genome further comprises a functionaldisruption of the endogenous KSE336; and

[0171] a mammalian cell whose genome comprises a recombinant KSE336operatively linked to an expression control sequence effective toexpress said gene in brain and pancreas cells, cells derived from brainand pancreas, or brain and pancreas progenitor cells.

[0172] Database

[0173] The present invention also relates to electronic forms ofpolynucleotides, polypeptides, etc., of the present invention, includingcomputer-readable medium (e.g., magnetic, optical, etc., stored in anysuitable format, such as flat files or hierarchical files) whichcomprise such sequences, or fragments thereof, e-commerce-related means,etc. Along these lines, the present invention relates to methods ofretrieving gene sequences from a computer-readable medium, comprising,one or more of the following steps in any effective order, e.g.,selecting a cell or gene expression profile, e.g., a profile thatspecifies that said gene is differentially expressed in brain andpancreas, and retrieving said differentially expressed gene sequences,where the gene sequences consist of the genes represented by SEQ ID NO 1and 2.

[0174] A “gene expression profile” means the list of tissues, cells,etc., in which a defined gene is expressed (i.e, transcribed and/ortranslated). A “cell expression profile” means the genes which areexpressed in the particular cell type. The profile can be a list of thetissues in which the gene is expressed, but can include additionalinformation as well, including level of expression (e.g., a quantity ascompared or normalized to a control gene), and information on temporal(e.g., at what point in the cell-cycle or developmental program) andspatial expression. By the phrase “selecting a gene or cell expressionprofile,” it is meant that a user decides what type of gene or cellexpression pattern he is interested in retrieving, e.g., he may requirethat the gene is differentially expressed in a tissue, or he may requirethat the gene is not expressed in blood, but must be expressed in brainand pancreas. Any pattern of expression preferences may be selected. Theselecting can be performed by any effective method. In general,“selecting” refers to the process in which a user forms a query that isused to search a database of gene expression profiles. The step ofretrieving involves searching for results in a database that correspondto the query set forth in the selecting step. Any suitable algorithm canbe utilized to perform the search query, including algorithms that lookfor matches, or that perform optimization between query and data. Thedatabase is information that has been stored in an appropriate storagemedium, having a suitable computer-readable format. Once results areretrieved, they can be displayed in any suitable format, such as HTML.

[0175] For instance, the user may be interested in identifying genesthat are differentially expressed in a brain and pancreas. He may notcare whether small amounts of expression occur in other tissues, as longas such genes are not expressed in peripheral blood lymphocytes. A queryis formed by the user to retrieve the set of genes from the databasehaving the desired gene or cell expression profile. Once the query isinputted into the system, a search algorithm is used to interrogate thedatabase, and retrieve results.

[0176] Advertising, Licensing, etc., Methods

[0177] The present invention also relates to methods of advertising,licensing, selling, purchasing, brokering, etc., genes, polynucleotides,specific-binding partners, antibodies, etc., of the present invention.Methods can comprises, e.g., displaying a KSE336 gene, KSE336polypeptide, or antibody specific for KSE336 in a printed orcomputer-readable medium (e.g., on the Web or Internet), accepting anoffer to purchase said gene, polypeptide, or antibody.

[0178] Other

[0179] A polynucleotide, probe, polypeptide, antibody, specific-bindingpartner, etc., according to the present invention can be isolated. Theterm “isolated” means that the material is in a form in which it is notfound in its original environment or in nature, e.g., more concentrated,more purified, separated from component, etc. An isolated polynucleotideincludes, e.g., a polynucleotide having the sequenced separated from thechromosomal DNA found in a living animal, e.g., as the complete gene, atranscript, or a cDNA. This polynucleotide can be part of a vector orinserted into a chromosome (by specific gene-targeting or by randomintegration at a position other than its normal position) and still beisolated in that it is not in a form that is found in its naturalenvironment. A polynucleotide, polypeptide, etc., of the presentinvention can also be substantially purified. By substantially purified,it is meant that polynucleotide or polypeptide is separated and isessentially free from other polynucleotides or polypeptides, i.e., thepolynucleotide or polypeptide is the primary and active constituent. Apolynucleotide can also be a recombinant molecule. By “recombinant,” itis meant that the polynucleotide is an arrangement or form which doesnot occur in nature. For instance, a recombinant molecule comprising apromoter sequence would not encompass the naturally-occurring gene, butwould include the promoter operably linked to a coding sequence notassociated with it in nature, e.g., a reporter gene, or a truncation ofthe normal coding sequence.

[0180] The term “marker” is used herein to indicate a means fordetecting or labeling a target. A marker can be a polynucleotide(usually referred to as a “probe”), polypeptide (e.g., an antibodyconjugated to a detectable label), PNA, or any effective material.

[0181] The term “consisting essentially” indicates that a compositionhas ingredients that are specifically identified in the claim but otheringredients may also be present, although not specifically identified inthe claim, so long as those other unlisted ingredients do not have amaterial effect on the basic and novel characteristics of thecomposition.

[0182] The topic headings set forth above are meant as guidance wherecertain information can be found in the application, but are notintended to be the only source in the application where information onsuch topic can be found.

[0183] Reference Materials

[0184] For other aspects of the polynucleotides, reference is made tostandard textbooks of molecular biology. See, e.g., Hames et al.,Polynucleotide Hybridization, IL Press, 1985; Davis et al., BasicMethods in Molecular Biology, Elsevir Sciences Publishing, Inc., NewYork, 1986; Sambrook et al., Molecular Cloning, CSH Press, 1989; Howe,Gene Cloning and Manipulation, Cambridge University Press, 1995; Ausubelet al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc.,1994-1998. The preceding description, utilize the present invention toits fullest extent. The preceding preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limiting theremainder of the disclosure in any way whatsoever. The entire disclosureof all applications, patents and publications, cited above and in thefigures are incorporated by reference in their entirety. TABLE 1 SEQStart End Nucleotide Sequence ID 34 84CAACTCCTATCTAAATTTCACCGGCATTGTTTGCAG 7 AGGCAGGGAAAGGG 441 491ACTAAAAATAAAAAAAAATTAGCCGGGCGTGGTGGC 8 GGGCACCTGTAGTC 1044 1094GTGTTCTCTTTATATATTGCTGGAATTGATTTGATG 9 TTTTGTTAAGGGAT 2136 2186CTACATTCAGCTAAAAATGTCTGCTGTCCCCACTCA 10 CAGCAGCAGCAGCG 3226 3276GCCCTGCAGGGTAAAACCCCCGTCCAGGGCAGCCAT 11 CTGCACCCCCTCGC 3301 23351CGGGATGCGCCTTAATGGCGGGTCGGGCGGCAGCGG 12 GAGCTCTGCTGCCT 3848 3898CTGGGGGCGCGGGGCGCGGGGCGCGGGCCTCGGCGG 13 CGGCGGCGGCGGCG 3860 3910GGCGCGGGGCGCGGGCCTCGGCGGCGGCGGCGGCGG 14 CGGCGGCGGAAGCC

[0185] TABLE 2 Type of Clone seq Genomic Seq. Clone name Polymorphism(Pos/nt) (Accn#, nt) FB1620G06/ Substitution  92, C NT_024164, TKSE336-1 Substitution 1248, G NT_024164, A Substitution 1340, CNT_024164, T Substitution 1353, C NT_024164, G Insertion 1370, CNT_024164, * Insertion 1443, G NT_024164, * Substitution 1615-1617, GAGNT_024164, AGT Deletion 2026-2027, * NT_024164, G Substitution 2072, CNT_024164, A Insertion 2206, G NT_024164, * Substitution 2473, CNT_024164, T Insertion 2607, C NT_024164, * Substitution 2611, TNT_024164, C Deletion 2648-2649, * NT_024164, G AB1138D11/ Substitution1467, C NT_024164, T KSE336-2 Substitution 1480, C NT_024164, GInsertion 1497, C NT_024164, * Insertion 1570, G NT_024164, *Substitution 1742-1744, GAG NT_024164, AGT Deletion 2153-2154, *NT_024164, G Substitution 2199, C NT_024164, A Insertion 2333, GNT_024164, * Substitution 2600, C NT_024164, T Insertion 2735, CNT_024164, * Deletion 2775-2776, * NT_024164, G Insertion 3143-3144, CTNT_024164, ** Substitution 3197, C NT_024164, T Insertion 3211, GNT_024164, *

[0186]

1 18 1 2908 DNA Homo sapiens CDS (106)..(2112) 1 ggccgggtcg gcgcggacggcactcggcgg acgcgggcgg acgctgggcg gcccctccct 60 gcccgcgcgc ccgggcgcccctggccggcg ccgggcccca gagcg atg aca tcg acg 117 Met Thr Ser Thr 1 gggaag gac ggc ggc gcg cag cac gcg cag tat gtt ggg ccc tac cgg 165 Gly LysAsp Gly Gly Ala Gln His Ala Gln Tyr Val Gly Pro Tyr Arg 5 10 15 20 ctggag aag acg ctg ggc aag ggg cag aca ggt ctg gtg aag ctg ggg 213 Leu GluLys Thr Leu Gly Lys Gly Gln Thr Gly Leu Val Lys Leu Gly 25 30 35 gtt cactgc gtc acc tgc cag aag gtg gcc atc aag atc gtc aac cgt 261 Val His CysVal Thr Cys Gln Lys Val Ala Ile Lys Ile Val Asn Arg 40 45 50 gag aag ctcagc gag tcg gtg ctg atg aag gtg gag cgg gag atc gcg 309 Glu Lys Leu SerGlu Ser Val Leu Met Lys Val Glu Arg Glu Ile Ala 55 60 65 atc ctg aag ctcatt gag cac ccc cac gtc cta aag ctg cac gac gtt 357 Ile Leu Lys Leu IleGlu His Pro His Val Leu Lys Leu His Asp Val 70 75 80 tat gaa aac aaa aaatat ttg tac ctg gtg cta gaa cac gtg tca ggt 405 Tyr Glu Asn Lys Lys TyrLeu Tyr Leu Val Leu Glu His Val Ser Gly 85 90 95 100 ggt gag ctc ttc gactac ctg gtg aag aag ggg agg ctg acg cct aag 453 Gly Glu Leu Phe Asp TyrLeu Val Lys Lys Gly Arg Leu Thr Pro Lys 105 110 115 gag gct cgg aag ttcttc cgg cag atc atc tct gcg ctg gac ttc tgc 501 Glu Ala Arg Lys Phe PheArg Gln Ile Ile Ser Ala Leu Asp Phe Cys 120 125 130 cac agc cac tcc atatgc cac agg gat ctg aaa cct gaa aac ctc ctg 549 His Ser His Ser Ile CysHis Arg Asp Leu Lys Pro Glu Asn Leu Leu 135 140 145 ctg gac gag aag aacaac atc cgc atc gca gac ttt ggc atg gcg tcc 597 Leu Asp Glu Lys Asn AsnIle Arg Ile Ala Asp Phe Gly Met Ala Ser 150 155 160 ctg cag gtt ggc gacagc ctg ttg gag acc agc tgt ggg tcc ccc cac 645 Leu Gln Val Gly Asp SerLeu Leu Glu Thr Ser Cys Gly Ser Pro His 165 170 175 180 tac gcc tgc cccgag gtg atc cgg ggg gag aag tat gac ggc cgg aag 693 Tyr Ala Cys Pro GluVal Ile Arg Gly Glu Lys Tyr Asp Gly Arg Lys 185 190 195 gcg gac gtg tggagc tgc ggc gtc atc ctg ttc gcc ttg ctg gtg ggg 741 Ala Asp Val Trp SerCys Gly Val Ile Leu Phe Ala Leu Leu Val Gly 200 205 210 gct ctg ccc ttcgac gat gac aac ttg cga cag ctg ctg gag aag gtg 789 Ala Leu Pro Phe AspAsp Asp Asn Leu Arg Gln Leu Leu Glu Lys Val 215 220 225 aag cgg ggc gtgttc cac atg ccg cac ttt atc ccg ccc gac tgc cag 837 Lys Arg Gly Val PheHis Met Pro His Phe Ile Pro Pro Asp Cys Gln 230 235 240 agt ctg cta cggggc atg atc gag gtg gac gcc gca cgc cgc ctc acg 885 Ser Leu Leu Arg GlyMet Ile Glu Val Asp Ala Ala Arg Arg Leu Thr 245 250 255 260 cta gag cacatt cag aaa cac ata tgg tat ata ggg ggc aag aat gag 933 Leu Glu His IleGln Lys His Ile Trp Tyr Ile Gly Gly Lys Asn Glu 265 270 275 ccc gaa ccagag cag ccc att cct cgc aag gtg cag atc cgc tcg ctg 981 Pro Glu Pro GluGln Pro Ile Pro Arg Lys Val Gln Ile Arg Ser Leu 280 285 290 ccc agc ctggag gac atc gac ccc gac gtg ctg gac agc atg cac tca 1029 Pro Ser Leu GluAsp Ile Asp Pro Asp Val Leu Asp Ser Met His Ser 295 300 305 ctg ggc tgcttc cga gac cgc aac aag ctg ctg cag gac ctg ctg tcc 1077 Leu Gly Cys PheArg Asp Arg Asn Lys Leu Leu Gln Asp Leu Leu Ser 310 315 320 gag gag gagaac cag gag aag atg att tac ttc ctc ctc ctg gac cgg 1125 Glu Glu Glu AsnGln Glu Lys Met Ile Tyr Phe Leu Leu Leu Asp Arg 325 330 335 340 aaa gaaagg tac ccg agc cag gag gat gag gac ctg ccc ccc cgg aac 1173 Lys Glu ArgTyr Pro Ser Gln Glu Asp Glu Asp Leu Pro Pro Arg Asn 345 350 355 gag atagac cct ccc cgg aag cgt gtg gac tcc ccg atg ctg aac cgg 1221 Glu Ile AspPro Pro Arg Lys Arg Val Asp Ser Pro Met Leu Asn Arg 360 365 370 cac ggcaag cgg cgg cca gaa cgc aag tcc atg gag gtg ctc agc gtg 1269 His Gly LysArg Arg Pro Glu Arg Lys Ser Met Glu Val Leu Ser Val 375 380 385 acg gacggc ggc tcc ccg gtg cct gcg cgg cgg gcc att gag atg gcc 1317 Thr Asp GlyGly Ser Pro Val Pro Ala Arg Arg Ala Ile Glu Met Ala 390 395 400 cag cacggc cag agg tct cgg tcc atc agc ggt gcc tcc tca ggc ctt 1365 Gln His GlyGln Arg Ser Arg Ser Ile Ser Gly Ala Ser Ser Gly Leu 405 410 415 420 tccacc agc cca ctc agc agc ccc cgg gtg acc cct cac ccc tca cca 1413 Ser ThrSer Pro Leu Ser Ser Pro Arg Val Thr Pro His Pro Ser Pro 425 430 435 aggggc agt ccc ctc ccc acc ccc aag ggg aca cct gtc cac acg cca 1461 Arg GlySer Pro Leu Pro Thr Pro Lys Gly Thr Pro Val His Thr Pro 440 445 450 aaggag agc ccg gct ggc acg ccc aac ccc acg ccc ccg tcc agc ccc 1509 Lys GluSer Pro Ala Gly Thr Pro Asn Pro Thr Pro Pro Ser Ser Pro 455 460 465 agcgtc gga ggg gtg ccc tgg agg gcg cgg ctc aac tcc atc aag aac 1557 Ser ValGly Gly Val Pro Trp Arg Ala Arg Leu Asn Ser Ile Lys Asn 470 475 480 agcttt ctg ggc tca ccc cgc ttc cac cgc cgg aaa ctg caa gtt ccg 1605 Ser PheLeu Gly Ser Pro Arg Phe His Arg Arg Lys Leu Gln Val Pro 485 490 495 500acg ccg gag gag atg tcc aac ctg aca cca gag tcg tcc cca gag ctg 1653 ThrPro Glu Glu Met Ser Asn Leu Thr Pro Glu Ser Ser Pro Glu Leu 505 510 515gcg aag aag tcc tgg ttt ggg aac ttc atc agc ctg gag aag gag gag 1701 AlaLys Lys Ser Trp Phe Gly Asn Phe Ile Ser Leu Glu Lys Glu Glu 520 525 530cag atc ttc gtg gtc atc aaa gac aaa cct ctg agc tcc atc aag gct 1749 GlnIle Phe Val Val Ile Lys Asp Lys Pro Leu Ser Ser Ile Lys Ala 535 540 545gac atc gtg cac gcc ttc ctg tcg att ccc agt ctc agc cac agc gtc 1797 AspIle Val His Ala Phe Leu Ser Ile Pro Ser Leu Ser His Ser Val 550 555 560atc tcc caa acg agc ttc cgg gcc gag tac aag gcc acg ggg ggg cca 1845 IleSer Gln Thr Ser Phe Arg Ala Glu Tyr Lys Ala Thr Gly Gly Pro 565 570 575580 gcc gtg ttc cag aag ccg gtc aag ttc cag gtt gat atc acc tac acg 1893Ala Val Phe Gln Lys Pro Val Lys Phe Gln Val Asp Ile Thr Tyr Thr 585 590595 gag ggt ggg gag gcg cag aag gag aac ggc atc tac tcc gtc acc ttc 1941Glu Gly Gly Glu Ala Gln Lys Glu Asn Gly Ile Tyr Ser Val Thr Phe 600 605610 acc ctg ctc tca ggc ccc agc cgt cgc ttc aag agg gtg gtg gag acc 1989Thr Leu Leu Ser Gly Pro Ser Arg Arg Phe Lys Arg Val Val Glu Thr 615 620625 atc cag gcc cag ctg ctg agc aca cac gac ccg cct gcg gcc cag cac 2037Ile Gln Ala Gln Leu Leu Ser Thr His Asp Pro Pro Ala Ala Gln His 630 635640 ttg tca gac acc act aac tgt atg gaa atg atg acg ggg cgg ctt tcc 2085Leu Ser Asp Thr Thr Asn Cys Met Glu Met Met Thr Gly Arg Leu Ser 645 650655 660 aaa tgt gga att atc ccg aaa agt taa catgtcacct ccacgaggcc 2132Lys Cys Gly Ile Ile Pro Lys Ser 665 atcctctgtg accgaaggca gctgctgcggacccgccctc cctccgctcc tgctgttgct 2192 gccgggcagt gaggcccagc ccagcgccccgtccaccccg cggcagctcc tcgcctcact 2252 ccgcacggcc cgtgggagga aggccaggctcgggggagcc tcctccagcc cggccgaccc 2312 ggactcccgg tcacctgacc cctcagcaagaacagcctgc ctggtggcct tctggggcca 2372 ggacccctgg tgggcaacgt agccacaggaacaggccccg tccaccgcct ccacgccgca 2432 cctggaggcc tcctcgcagg cccgtgccccgcctccctgc cgcgccgcct ccgtgtagtc 2492 ttggcctcct caggctgcct cccgtcctctcgtctcaccc gcgcctccct tgcctcatct 2552 ggggcggctg tgggctctgg cgctcctctctggctgaggt ggaaacagag acaccctgtg 2612 gcaccagagc cttcccagca ggccaggccgctgggctggg atcagtgtta tttatttgcc 2672 gttttaattt atggattctc cgcacctctgttcagggaag ggcggcggcc acatcccctg 2732 ccgtctgcgt gtctcaggca gtgggggggctggggccagg gcgccctctg aggacagagc 2792 tggtggggcg cgggggggct ggcgagctactgtaaacttt aaagaattcc tgcaagatat 2852 ttttataaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaa 2908 2 668 PRT Homo sapiens 2 Met Thr SerThr Gly Lys Asp Gly Gly Ala Gln His Ala Gln Tyr Val 1 5 10 15 Gly ProTyr Arg Leu Glu Lys Thr Leu Gly Lys Gly Gln Thr Gly Leu 20 25 30 Val LysLeu Gly Val His Cys Val Thr Cys Gln Lys Val Ala Ile Lys 35 40 45 Ile ValAsn Arg Glu Lys Leu Ser Glu Ser Val Leu Met Lys Val Glu 50 55 60 Arg GluIle Ala Ile Leu Lys Leu Ile Glu His Pro His Val Leu Lys 65 70 75 80 LeuHis Asp Val Tyr Glu Asn Lys Lys Tyr Leu Tyr Leu Val Leu Glu 85 90 95 HisVal Ser Gly Gly Glu Leu Phe Asp Tyr Leu Val Lys Lys Gly Arg 100 105 110Leu Thr Pro Lys Glu Ala Arg Lys Phe Phe Arg Gln Ile Ile Ser Ala 115 120125 Leu Asp Phe Cys His Ser His Ser Ile Cys His Arg Asp Leu Lys Pro 130135 140 Glu Asn Leu Leu Leu Asp Glu Lys Asn Asn Ile Arg Ile Ala Asp Phe145 150 155 160 Gly Met Ala Ser Leu Gln Val Gly Asp Ser Leu Leu Glu ThrSer Cys 165 170 175 Gly Ser Pro His Tyr Ala Cys Pro Glu Val Ile Arg GlyGlu Lys Tyr 180 185 190 Asp Gly Arg Lys Ala Asp Val Trp Ser Cys Gly ValIle Leu Phe Ala 195 200 205 Leu Leu Val Gly Ala Leu Pro Phe Asp Asp AspAsn Leu Arg Gln Leu 210 215 220 Leu Glu Lys Val Lys Arg Gly Val Phe HisMet Pro His Phe Ile Pro 225 230 235 240 Pro Asp Cys Gln Ser Leu Leu ArgGly Met Ile Glu Val Asp Ala Ala 245 250 255 Arg Arg Leu Thr Leu Glu HisIle Gln Lys His Ile Trp Tyr Ile Gly 260 265 270 Gly Lys Asn Glu Pro GluPro Glu Gln Pro Ile Pro Arg Lys Val Gln 275 280 285 Ile Arg Ser Leu ProSer Leu Glu Asp Ile Asp Pro Asp Val Leu Asp 290 295 300 Ser Met His SerLeu Gly Cys Phe Arg Asp Arg Asn Lys Leu Leu Gln 305 310 315 320 Asp LeuLeu Ser Glu Glu Glu Asn Gln Glu Lys Met Ile Tyr Phe Leu 325 330 335 LeuLeu Asp Arg Lys Glu Arg Tyr Pro Ser Gln Glu Asp Glu Asp Leu 340 345 350Pro Pro Arg Asn Glu Ile Asp Pro Pro Arg Lys Arg Val Asp Ser Pro 355 360365 Met Leu Asn Arg His Gly Lys Arg Arg Pro Glu Arg Lys Ser Met Glu 370375 380 Val Leu Ser Val Thr Asp Gly Gly Ser Pro Val Pro Ala Arg Arg Ala385 390 395 400 Ile Glu Met Ala Gln His Gly Gln Arg Ser Arg Ser Ile SerGly Ala 405 410 415 Ser Ser Gly Leu Ser Thr Ser Pro Leu Ser Ser Pro ArgVal Thr Pro 420 425 430 His Pro Ser Pro Arg Gly Ser Pro Leu Pro Thr ProLys Gly Thr Pro 435 440 445 Val His Thr Pro Lys Glu Ser Pro Ala Gly ThrPro Asn Pro Thr Pro 450 455 460 Pro Ser Ser Pro Ser Val Gly Gly Val ProTrp Arg Ala Arg Leu Asn 465 470 475 480 Ser Ile Lys Asn Ser Phe Leu GlySer Pro Arg Phe His Arg Arg Lys 485 490 495 Leu Gln Val Pro Thr Pro GluGlu Met Ser Asn Leu Thr Pro Glu Ser 500 505 510 Ser Pro Glu Leu Ala LysLys Ser Trp Phe Gly Asn Phe Ile Ser Leu 515 520 525 Glu Lys Glu Glu GlnIle Phe Val Val Ile Lys Asp Lys Pro Leu Ser 530 535 540 Ser Ile Lys AlaAsp Ile Val His Ala Phe Leu Ser Ile Pro Ser Leu 545 550 555 560 Ser HisSer Val Ile Ser Gln Thr Ser Phe Arg Ala Glu Tyr Lys Ala 565 570 575 ThrGly Gly Pro Ala Val Phe Gln Lys Pro Val Lys Phe Gln Val Asp 580 585 590Ile Thr Tyr Thr Glu Gly Gly Glu Ala Gln Lys Glu Asn Gly Ile Tyr 595 600605 Ser Val Thr Phe Thr Leu Leu Ser Gly Pro Ser Arg Arg Phe Lys Arg 610615 620 Val Val Glu Thr Ile Gln Ala Gln Leu Leu Ser Thr His Asp Pro Pro625 630 635 640 Ala Ala Gln His Leu Ser Asp Thr Thr Asn Cys Met Glu MetMet Thr 645 650 655 Gly Arg Leu Ser Lys Cys Gly Ile Ile Pro Lys Ser 660665 3 3364 DNA Homo sapiens CDS (482)..(2239) 3 ctcgacgagg cggaggcgtcgccgcgggcc aggcctcgga ctgccgcgtc ggagtggacg 60 cggggggcgg cggcgcgggcggacgcgggc ggcgcgaagc agcggggccc gcgggggcgc 120 cccggccggg tcggcgcggacggcactcgg cggacgcggg cggacgctgg gcggcccctc 180 cctgcccgcg cgcccgggcgcccctggccg gcgctgggcc ccagagcgat gacatcgacg 240 gggaaggacg gcggcgcgcagcacgcgcag tatgttgggc cctaccggct ggagaagacg 300 ctgggcaagg ggcagacaggtctggtgaag ctgggggttc actgcgtcac ctgccagaag 360 gtggccatca agatcgtcaaccgtgagaag ctcagcgagt cggtgctgat gaaggtggag 420 cgggagatcg cgatcctgaagctcattgag cacccccacg tcctaaagct gcacgacgtt 480 t atg aaa aca aaa aatatt tgt agg tac ctg gtg cta gaa cac gtg tca 529 Met Lys Thr Lys Asn IleCys Arg Tyr Leu Val Leu Glu His Val Ser 1 5 10 15 ggt ggt gag ctc ttcgac tac ctg gtg aag aag ggg agg ctg acg cct 577 Gly Gly Glu Leu Phe AspTyr Leu Val Lys Lys Gly Arg Leu Thr Pro 20 25 30 aag gag gct cgg aag ttcttc cgg cag atc atc tct gcg ctg gac ttc 625 Lys Glu Ala Arg Lys Phe PheArg Gln Ile Ile Ser Ala Leu Asp Phe 35 40 45 tgc cac agc cac tcc ata tgccac agg gat ctg aaa cct gaa aac ctc 673 Cys His Ser His Ser Ile Cys HisArg Asp Leu Lys Pro Glu Asn Leu 50 55 60 ctg ctg gac gag aag aac aac atccgc atc gca gac ttt ggc atg gcg 721 Leu Leu Asp Glu Lys Asn Asn Ile ArgIle Ala Asp Phe Gly Met Ala 65 70 75 80 tcc ctg cag gtt ggc gac agc ctgttg gag acc agc tgt ggg tcc ccc 769 Ser Leu Gln Val Gly Asp Ser Leu LeuGlu Thr Ser Cys Gly Ser Pro 85 90 95 cac tac gcc tgc ccc gag gtg atc cggggg gag aag tat gac ggc cgg 817 His Tyr Ala Cys Pro Glu Val Ile Arg GlyGlu Lys Tyr Asp Gly Arg 100 105 110 aag gcg gac gtg tgg agc tgc ggc gtcatc ctg ttc gcc ttg ctg gtg 865 Lys Ala Asp Val Trp Ser Cys Gly Val IleLeu Phe Ala Leu Leu Val 115 120 125 ggg gct ctg ccc ttc gac gat gac aacttg cga cag ctg ctg gag aag 913 Gly Ala Leu Pro Phe Asp Asp Asp Asn LeuArg Gln Leu Leu Glu Lys 130 135 140 gtg aag cgg ggc gtg ttc cac atg ccgcac ttt atc ccg ccc gac tgc 961 Val Lys Arg Gly Val Phe His Met Pro HisPhe Ile Pro Pro Asp Cys 145 150 155 160 cag agt ctg cta cgg ggc atg atcgag gtg gac gcc gca cgc cgc ctc 1009 Gln Ser Leu Leu Arg Gly Met Ile GluVal Asp Ala Ala Arg Arg Leu 165 170 175 acg cta gag cac att cag aaa cacata tgg tat ata ggg ggc aag aat 1057 Thr Leu Glu His Ile Gln Lys His IleTrp Tyr Ile Gly Gly Lys Asn 180 185 190 gag ccc gaa cca gag cag ccc attcct cgc aag gtg cag atc cgc tcg 1105 Glu Pro Glu Pro Glu Gln Pro Ile ProArg Lys Val Gln Ile Arg Ser 195 200 205 ctg ccc agc ctg gag gac atc gacccc gac gtg ctg gac agc atg cac 1153 Leu Pro Ser Leu Glu Asp Ile Asp ProAsp Val Leu Asp Ser Met His 210 215 220 tca ctg ggc tgc ttc cga gac cgcaac aag ctg ctg cag gac ctg ctg 1201 Ser Leu Gly Cys Phe Arg Asp Arg AsnLys Leu Leu Gln Asp Leu Leu 225 230 235 240 tcc gag gag gag aac cag gagaag atg att tac ttc ctc ctc ctg gac 1249 Ser Glu Glu Glu Asn Gln Glu LysMet Ile Tyr Phe Leu Leu Leu Asp 245 250 255 cgg aaa gaa agg tac ccg agccag gag gat gag gac ctg ccc ccc cgg 1297 Arg Lys Glu Arg Tyr Pro Ser GlnGlu Asp Glu Asp Leu Pro Pro Arg 260 265 270 aac gag ata gac cct ccc cggaag cgt gtg gac tcc ccg atg ctg aac 1345 Asn Glu Ile Asp Pro Pro Arg LysArg Val Asp Ser Pro Met Leu Asn 275 280 285 cgg cac ggc aag cgg cgg ccagaa cgc aaa tcc atg gag gtg ctc agc 1393 Arg His Gly Lys Arg Arg Pro GluArg Lys Ser Met Glu Val Leu Ser 290 295 300 gtg acg gac ggc ggc tcc ccggtg cct gcg cgg cgg gcc att gag atg 1441 Val Thr Asp Gly Gly Ser Pro ValPro Ala Arg Arg Ala Ile Glu Met 305 310 315 320 gcc cag cac ggc cag aggtct cgg tcc atc agc ggt gcc tcc tca ggc 1489 Ala Gln His Gly Gln Arg SerArg Ser Ile Ser Gly Ala Ser Ser Gly 325 330 335 ctt tcc acc agc cca ctcagc agc ccc cgg gtg acc cct cac ccc tca 1537 Leu Ser Thr Ser Pro Leu SerSer Pro Arg Val Thr Pro His Pro Ser 340 345 350 cca agg ggc agt ccc ctcccc acc ccc aag ggg aca cct gtc cac acg 1585 Pro Arg Gly Ser Pro Leu ProThr Pro Lys Gly Thr Pro Val His Thr 355 360 365 cca aag gag agc ccg gctggc acg ccc aac ccc acg ccc ccg tcc agc 1633 Pro Lys Glu Ser Pro Ala GlyThr Pro Asn Pro Thr Pro Pro Ser Ser 370 375 380 ccc agc gtc gga ggg gtgccc tgg agg gcg cgg ctc aac tcc atc aag 1681 Pro Ser Val Gly Gly Val ProTrp Arg Ala Arg Leu Asn Ser Ile Lys 385 390 395 400 aac agc ttt ctg ggctca ccc cgc ttc cac cgc cgg aaa ctg caa gtt 1729 Asn Ser Phe Leu Gly SerPro Arg Phe His Arg Arg Lys Leu Gln Val 405 410 415 ccg acg ccg gag gagatg tcc aac ctg aca cca gag tcg tcc cca gag 1777 Pro Thr Pro Glu Glu MetSer Asn Leu Thr Pro Glu Ser Ser Pro Glu 420 425 430 ctg gcg aag aag tcctgg ttt ggg aac ttc atc agc ctg gag aag gag 1825 Leu Ala Lys Lys Ser TrpPhe Gly Asn Phe Ile Ser Leu Glu Lys Glu 435 440 445 gag cag atc ttc gtggtc atc aaa gac aaa cct ctg agc tcc atc aag 1873 Glu Gln Ile Phe Val ValIle Lys Asp Lys Pro Leu Ser Ser Ile Lys 450 455 460 gct gac atc gtg cacgcc ttc ctg tcg att ccc agt ctc agc cac agc 1921 Ala Asp Ile Val His AlaPhe Leu Ser Ile Pro Ser Leu Ser His Ser 465 470 475 480 gtc atc tcc caaacg agc ttc cgg gcc gag tac aag gcc acg ggg ggg 1969 Val Ile Ser Gln ThrSer Phe Arg Ala Glu Tyr Lys Ala Thr Gly Gly 485 490 495 cca gcc gtg ttccag aag ccg gtc aag ttc cag gtt gat atc acc tac 2017 Pro Ala Val Phe GlnLys Pro Val Lys Phe Gln Val Asp Ile Thr Tyr 500 505 510 acg gag ggt ggggag gcg cag aag gag aac ggc atc tac tcc gtc acc 2065 Thr Glu Gly Gly GluAla Gln Lys Glu Asn Gly Ile Tyr Ser Val Thr 515 520 525 ttc acc ctg ctctca ggc ccc agc cgt cgc ttc aag agg gtg gtg gag 2113 Phe Thr Leu Leu SerGly Pro Ser Arg Arg Phe Lys Arg Val Val Glu 530 535 540 acc atc cag gcccag ctg ctg agc aca cac gac ccg cct gcg gcc cag 2161 Thr Ile Gln Ala GlnLeu Leu Ser Thr His Asp Pro Pro Ala Ala Gln 545 550 555 560 cac ttg tcagac acc act aac tgt atg gaa atg atg acg ggg cgg ctt 2209 His Leu Ser AspThr Thr Asn Cys Met Glu Met Met Thr Gly Arg Leu 565 570 575 tcc aaa tgtgga att atc ccg aaa agt taa catgtcacct ccacgaggcc 2259 Ser Lys Cys GlyIle Ile Pro Lys Ser 580 585 atcctctgtg accgaaggca gctgctgcgg acccgccctccctccgctcc tgctgttgct 2319 gccgggcagt gaggcccagc ccagcgcccc gtccaccccgcggcagctcc tcgcctcact 2379 ccgcacggcc cgtgggagga aggccaggct cgggggagcctcctccagcc cggccgaccc 2439 ggactcccgg tcacctgacc cctcagcaag aacagcctgcctggtggcct tctggggcca 2499 ggacccctgg tgggcaacgt agccacagga acaggccccgtccaccgcct ccacgccgca 2559 cctggaggcc tcctcgcagg cccgtgcccc gcctccctgccgcgccgcct ccgtgtagtc 2619 ttggcctcct caggctgcct cccgtcctct cgtctcacccgcgcctccct tgcctcatct 2679 ggggcggctg tgggctctgg cgctcctctc tggctgaggtggaaacagag acaccctgcg 2739 gcaccagagc cttcccagca ggccaggccg ctgggctgggatcagtgtta tttatttgcc 2799 gttttaattt atggattctc cgcacctctg ttcagggaagggcggcggcc acatcccctg 2859 ccgtctgcgt gtctcaggca gtgggggggc tggggccagggcgccctctg aggacagagc 2919 tggtggggcg cgggggggct ggcgagctac tgtaaactttaaagaattcc tgcaagatat 2979 ttttataaac ttttttttct tggtggtttt tggaaaagggtgtgggggtg ggggcgccgc 3039 tggggcaggg ccaggttttg tgttttagtc ccttgctcctgcttctttct acacacacat 3099 ctaaagacgg tgcggctcgc tctgtcatgg gttccgtctctctctgtgga gaagcagctc 3159 cacctctggg ggggctcggg gcagaggggc ggtgtctcgtagcgggcggc agcgccagcg 3219 cccctctgtc aggctggggc aatcttggtt ttgtgtccaaaggtgaaggg gtaggaggag 3279 ggccctcagc tggccctccc cacacacagg acggcaggggcactgtgagg cttttcttat 3339 taaaatgaaa aaaaaaaaaa aaaaa 3364 4 585 PRTHomo sapiens 4 Met Lys Thr Lys Asn Ile Cys Arg Tyr Leu Val Leu Glu HisVal Ser 1 5 10 15 Gly Gly Glu Leu Phe Asp Tyr Leu Val Lys Lys Gly ArgLeu Thr Pro 20 25 30 Lys Glu Ala Arg Lys Phe Phe Arg Gln Ile Ile Ser AlaLeu Asp Phe 35 40 45 Cys His Ser His Ser Ile Cys His Arg Asp Leu Lys ProGlu Asn Leu 50 55 60 Leu Leu Asp Glu Lys Asn Asn Ile Arg Ile Ala Asp PheGly Met Ala 65 70 75 80 Ser Leu Gln Val Gly Asp Ser Leu Leu Glu Thr SerCys Gly Ser Pro 85 90 95 His Tyr Ala Cys Pro Glu Val Ile Arg Gly Glu LysTyr Asp Gly Arg 100 105 110 Lys Ala Asp Val Trp Ser Cys Gly Val Ile LeuPhe Ala Leu Leu Val 115 120 125 Gly Ala Leu Pro Phe Asp Asp Asp Asn LeuArg Gln Leu Leu Glu Lys 130 135 140 Val Lys Arg Gly Val Phe His Met ProHis Phe Ile Pro Pro Asp Cys 145 150 155 160 Gln Ser Leu Leu Arg Gly MetIle Glu Val Asp Ala Ala Arg Arg Leu 165 170 175 Thr Leu Glu His Ile GlnLys His Ile Trp Tyr Ile Gly Gly Lys Asn 180 185 190 Glu Pro Glu Pro GluGln Pro Ile Pro Arg Lys Val Gln Ile Arg Ser 195 200 205 Leu Pro Ser LeuGlu Asp Ile Asp Pro Asp Val Leu Asp Ser Met His 210 215 220 Ser Leu GlyCys Phe Arg Asp Arg Asn Lys Leu Leu Gln Asp Leu Leu 225 230 235 240 SerGlu Glu Glu Asn Gln Glu Lys Met Ile Tyr Phe Leu Leu Leu Asp 245 250 255Arg Lys Glu Arg Tyr Pro Ser Gln Glu Asp Glu Asp Leu Pro Pro Arg 260 265270 Asn Glu Ile Asp Pro Pro Arg Lys Arg Val Asp Ser Pro Met Leu Asn 275280 285 Arg His Gly Lys Arg Arg Pro Glu Arg Lys Ser Met Glu Val Leu Ser290 295 300 Val Thr Asp Gly Gly Ser Pro Val Pro Ala Arg Arg Ala Ile GluMet 305 310 315 320 Ala Gln His Gly Gln Arg Ser Arg Ser Ile Ser Gly AlaSer Ser Gly 325 330 335 Leu Ser Thr Ser Pro Leu Ser Ser Pro Arg Val ThrPro His Pro Ser 340 345 350 Pro Arg Gly Ser Pro Leu Pro Thr Pro Lys GlyThr Pro Val His Thr 355 360 365 Pro Lys Glu Ser Pro Ala Gly Thr Pro AsnPro Thr Pro Pro Ser Ser 370 375 380 Pro Ser Val Gly Gly Val Pro Trp ArgAla Arg Leu Asn Ser Ile Lys 385 390 395 400 Asn Ser Phe Leu Gly Ser ProArg Phe His Arg Arg Lys Leu Gln Val 405 410 415 Pro Thr Pro Glu Glu MetSer Asn Leu Thr Pro Glu Ser Ser Pro Glu 420 425 430 Leu Ala Lys Lys SerTrp Phe Gly Asn Phe Ile Ser Leu Glu Lys Glu 435 440 445 Glu Gln Ile PheVal Val Ile Lys Asp Lys Pro Leu Ser Ser Ile Lys 450 455 460 Ala Asp IleVal His Ala Phe Leu Ser Ile Pro Ser Leu Ser His Ser 465 470 475 480 ValIle Ser Gln Thr Ser Phe Arg Ala Glu Tyr Lys Ala Thr Gly Gly 485 490 495Pro Ala Val Phe Gln Lys Pro Val Lys Phe Gln Val Asp Ile Thr Tyr 500 505510 Thr Glu Gly Gly Glu Ala Gln Lys Glu Asn Gly Ile Tyr Ser Val Thr 515520 525 Phe Thr Leu Leu Ser Gly Pro Ser Arg Arg Phe Lys Arg Val Val Glu530 535 540 Thr Ile Gln Ala Gln Leu Leu Ser Thr His Asp Pro Pro Ala AlaGln 545 550 555 560 His Leu Ser Asp Thr Thr Asn Cys Met Glu Met Met ThrGly Arg Leu 565 570 575 Ser Lys Cys Gly Ile Ile Pro Lys Ser 580 585 5213 DNA Homo sapiens CDS (1)..(213) 5 atg aca tcg acg ggg aag gac ggcggc gcg cag cac gcg cag tat gtt 48 Met Thr Ser Thr Gly Lys Asp Gly GlyAla Gln His Ala Gln Tyr Val 1 5 10 15 ggg ccc tac cgg ctg gag aag acgctg ggc aag ggg cag aca ggt ctg 96 Gly Pro Tyr Arg Leu Glu Lys Thr LeuGly Lys Gly Gln Thr Gly Leu 20 25 30 gtg aag ctg ggg gtt cac tgc gtc acctgc cag aag gtg gcc atc aag 144 Val Lys Leu Gly Val His Cys Val Thr CysGln Lys Val Ala Ile Lys 35 40 45 atc gtc aac cgt gag aag ctc agc gag tcggtg ctg atg aag gtg gag 192 Ile Val Asn Arg Glu Lys Leu Ser Glu Ser ValLeu Met Lys Val Glu 50 55 60 cgg gag atc gcg atc ctg aag 213 Arg Glu IleAla Ile Leu Lys 65 70 6 71 PRT Homo sapiens 6 Met Thr Ser Thr Gly LysAsp Gly Gly Ala Gln His Ala Gln Tyr Val 1 5 10 15 Gly Pro Tyr Arg LeuGlu Lys Thr Leu Gly Lys Gly Gln Thr Gly Leu 20 25 30 Val Lys Leu Gly ValHis Cys Val Thr Cys Gln Lys Val Ala Ile Lys 35 40 45 Ile Val Asn Arg GluLys Leu Ser Glu Ser Val Leu Met Lys Val Glu 50 55 60 Arg Glu Ile Ala IleLeu Lys 65 70 7 50 DNA Homo sapiens 7 caactcctat ctaaatttca ccggcattgtttgcagaggc agggaaaggg 50 8 50 DNA Homo sapiens 8 actaaaaata aaaaaaaattagccgggcgt ggtggcgggc acctgtagtc 50 9 50 DNA Homo sapiens 9 gtgttctctttatatattgc tggaattgat ttgatgtttt gttaagggat 50 10 50 DNA Homo sapiens 10ctacattcag ctaaaaatgt ctgctgtccc cactcacagc agcagcagcg 50 11 50 DNA Homosapiens 11 gccctgcagg gtaaaacccc cgtccagggc agccatctgc accccctcgc 50 1250 DNA Homo sapiens 12 cgggatgcgc cttaatggcg ggtcgggcgg cagcgggagctctgctgcct 50 13 50 DNA Homo sapiens 13 ctgggggcgc ggggcgcggg gcgcgggcctcggcggcggc ggcggcggcg 50 14 50 DNA Homo sapiens 14 ggcgcggggc gcgggcctcggcggcggcgg cggcggcggc ggcggaagcc 50 15 15 PRT Homo sapiens 15 His MetArg Ser Ala Met Ser Gly Leu His Leu Val Lys Arg Arg 1 5 10 15 16 7 PRTHomo sapiens 16 Leu Arg Arg Ala Ser Leu Gly 1 5 17 603 PRT Homo sapiens17 Leu Ile Glu His Pro His Val Leu Lys Leu His Asp Val Tyr Glu Asn 1 510 15 Lys Lys Tyr Leu Tyr Leu Val Leu Glu His Val Ser Gly Gly Glu Leu 2025 30 Phe Asp Tyr Leu Val Lys Lys Gly Arg Leu Thr Pro Lys Glu Ala Arg 3540 45 Lys Phe Phe Arg Gln Ile Ile Ser Ala Leu Asp Phe Cys His Ser His 5055 60 Ser Ile Cys His Arg Asp Leu Lys Pro Glu Asn Leu Leu Leu Asp Glu 6570 75 80 Lys Asn Asn Ile Arg Ile Ala Asp Phe Gly Met Ala Ser Leu Gln Val85 90 95 Gly Asp Ser Leu Leu Glu Thr Ser Cys Gly Ser Pro His Tyr Ala Cys100 105 110 Pro Glu Val Ile Arg Gly Glu Lys Tyr Asp Gly Arg Lys Ala AspVal 115 120 125 Trp Ser Cys Gly Val Ile Leu Phe Ala Leu Leu Val Gly AlaLeu Pro 130 135 140 Phe Asp Asp Asp Asn Leu Arg Gln Leu Leu Glu Lys ValLys Arg Gly 145 150 155 160 Val Phe His Met Pro His Phe Ile Pro Pro AspCys Gln Ser Leu Leu 165 170 175 Arg Gly Met Ser Glu Val Asp Ala Ala ArgArg Leu Thr Leu Glu His 180 185 190 Ile Gln Lys His Ile Trp Tyr Ile GlyGly Lys Asn Glu Pro Glu Pro 195 200 205 Glu Gln Pro Ile Pro Arg Lys ValGln Ile Arg Ser Leu Pro Ser Leu 210 215 220 Glu Asp Ile Asp Pro Asp ValLeu Asp Ser Met His Ser Leu Gly Cys 225 230 235 240 Phe Arg Asp Arg AsnLys Leu Leu Gln Asp Leu Leu Ser Glu Glu Glu 245 250 255 Asn Gln Glu LysMet Ile Tyr Phe Leu Leu Leu Asp Arg Lys Glu Arg 260 265 270 Tyr Pro SerGln Glu Asp Glu Asp Leu Pro Pro Arg Asn Glu Ile Asp 275 280 285 Pro ProArg Lys Arg Val Asp Ser Pro Met Leu Asn Arg His Gly Lys 290 295 300 ArgArg Pro Glu Arg Lys Ser Met Glu Val Leu Ser Val Thr Asp Gly 305 310 315320 Gly Ser Pro Val Pro Ala Arg Arg Ala Ile Glu Met Ala Gln His Gly 325330 335 Gln Arg Ser Arg Ser Ile Ser Gly Ala Ser Ser Gly Leu Ser Thr Ser340 345 350 Pro Leu Ser Ser Pro Arg Val Thr Pro His Pro Ser Pro Arg GlySer 355 360 365 Pro Leu Pro Thr Pro Lys Gly Thr Pro Val His Thr Pro LysGlu Ser 370 375 380 Pro Ala Gly Thr Pro Asn Pro Thr Pro Pro Ser Ser ProSer Val Gly 385 390 395 400 Gly Val Pro Trp Arg Ala Arg Leu Asn Ser IleLys Asn Ser Phe Leu 405 410 415 Gly Ser Pro Arg Phe His Arg Arg Lys LeuGln Val Pro Thr Pro Glu 420 425 430 Glu Met Ser Asn Leu Thr Pro Glu SerSer Pro Glu Leu Ala Lys Lys 435 440 445 Ser Trp Phe Gly Asn Phe Ile SerLeu Glu Lys Glu Glu Gln Ile Phe 450 455 460 Val Val Ile Lys Asp Lys ProLeu Ser Ser Ile Lys Ala Asp Ile Val 465 470 475 480 His Ala Phe Leu SerIle Pro Ser Leu Ser His Ser Val Ile Ser Gln 485 490 495 Thr Ser Phe ArgAla Glu Tyr Lys Ala Thr Gly Gly Pro Ala Val Phe 500 505 510 Gln Lys ProVal Lys Phe Gln Val Asp Ile Thr Tyr Thr Glu Gly Gly 515 520 525 Glu AlaGln Lys Glu Asn Gly Ile Tyr Ser Val Thr Phe Thr Leu Leu 530 535 540 SerGly Pro Ser Arg Arg Phe Lys Arg Val Val Glu Thr Ile Gln Ala 545 550 555560 Gln Leu Leu Ser Thr His Asp Pro Pro Ala Ala Gln His Leu Ser Glu 565570 575 Pro Pro Pro Pro Ala Pro Gly Leu Ser Trp Gly Ala Gly Leu Lys Gly580 585 590 Gln Lys Val Ala Thr Ser Tyr Glu Ser Ser Leu 595 600 18 149PRT Homo sapiens 18 Met Ser Asn Leu Thr Pro Glu Ser Ser Pro Glu Leu AlaLys Lys Ser 1 5 10 15 Trp Phe Gly Asn Phe Ile Ser Leu Glu Lys Glu GluGln Ile Phe Val 20 25 30 Val Ile Lys Asp Lys Pro Leu Ser Ser Ile Lys AlaAsp Ile Val His 35 40 45 Ala Phe Leu Ser Ile Pro Ser Leu Ser His Ser ValIle Ser Gln Thr 50 55 60 Ser Phe Arg Ala Glu Tyr Lys Ala Thr Gly Gly ProAla Val Phe Gln 65 70 75 80 Lys Pro Val Lys Phe Gln Val Asp Ile Thr TyrThr Glu Gly Gly Glu 85 90 95 Ala Gln Lys Glu Asn Gly Ile Tyr Ser Val ThrPhe Thr Leu Leu Ser 100 105 110 Gly Pro Ser Arg Arg Phe Lys Arg Val ValGlu Thr Ile Gln Ala Gln 115 120 125 Leu Leu Ser Thr His Asp Pro Leu ArgPro Ser Thr Cys Gln Thr Pro 130 135 140 Leu Thr Val Trp Lys 145

1. An isolated polynucleotide KSE336 which codes without interruptionfor an amino acid sequence set forth in SEQ ID NO 2 or SEQ ID NO 4, or acomplement thereto.
 2. An isolated polynucleotide KSE336 which has 99%or more sequence identity to a polynucleotide set forth in SEQ ID NO. 1or 3, or a complement thereto.
 3. An isolated polynucleotide of claim 1,which is SEQ ID NO
 1. 4. An isolated polynucleotide of claim 3, which isSEQ ID NO
 3. 5. An isolated polynucleotide of claim 1, which has SEQ IDNO 1 or SEQ IS NO 3, except for a polymorphism of Table
 1. 6. Anisolated polynucleotide consisting of: a polynucleotide fragment codingfor an amino acid sequence of SEQ ID NO. 6, a fragment thereof which isspecific for KSE336-1, or a complement thereto.
 7. An isolatedpolypeptide coded for by a polynucleotide of claim
 1. 8. A method oftreating a disease of brain or pancreas showing altered expression ofKSE336, comprising: administering to a subject in need thereof atherapeutic agent which is effective for regulating expression of saidKSE336 of claim
 1. 9. A method of claim 8, wherein said agent is anantibody or an antisense which is effective to inhibit translation ofsaid gene.
 10. A method of diagnosing a brain or pancreas diseaseassociated with abnormal KSE336, or susceptibility to said disease,comprising: assessing the expression of KSE336 of claim 1 in a tissuesample comprising pancreas cells, brain cells, or cells derived frompancreas or brain.
 11. A method of claim 10, wherein assessing is:measuring expression levels of said gene, determining the genomicstructure of said gene, determining the mRNA structure of transcriptsfrom said gene, or measuring the expression levels of polypeptide codedfor by said gene.
 12. A method of claim 1 1, further comprising:comparing said expression to the expression of said gene of a knownnormal tissue.
 13. A method of claim 10, wherein said assessing isperformed by: Northern blot analysis, polymerase chain reaction (PCR),reverse transcriptase PCR, RACE PCR, or in situ hybridization, and usinga polynucleotide probe having a sequence selected from SEQ ID NO 1 or acomplement thereto.
 14. A method of claim 10, wherein said disease isastrocytoma, meningioma, pancreatic adenocarcinoma, insulin-dependentdiabetes mellitus, Beckwith-Wiedemann syndrome, or congenitalhyperinsulinism
 15. A method of assessing a therapeutic or preventativeintervention in a subject having a brain or pancreas disease,comprising, determining the expression levels of KSE336 of claim 1 in atissue sample comprising brain and pancreas cells, or cells derived frombrain and pancreas.
 16. A method for identifying an agent that modulatesthe expression of KSE336 in brain or pancreas cells, cells derived frombrain and pancreas, or brain and pancreas progenitor cells, comprising,contacting a cell population with a test agent under conditionseffective for said test agent to modulate the expression of KSE336 ofclaim 1 in brain cells, pancreas cells, cells derived from brain orpancreas cells, or brain or pancreas progenitor cells, and determiningwhether said test agent modulates said KSE336.
 17. A method of claim 16,wherein said agent is an antisense polynucleotide to a targetpolynucleotide sequence selected from SEQ ID NO 1 which is effective toinhibit translation of said KSE336.
 18. A method of detectingpolymorphisms in KSE336 comprising: comparing the structure of: genomicDNA comprising all or part of KSE336, mRNA comprising all or part ofKSE336, cDNA comprising all or part of KSE336, or a polypeptidecomprising all or part of KSE336, with the structure of KSE336 of claim2.
 19. A method of claim 18, wherein said polymorphism is a nucleotidedeletion, substitution, inversion, or transposition.
 20. A method ofadvertising KSE336 for sale, commercial use, or licensing, comprising,displaying in a computer-readable medium a polynucleotide of claim 1,effective specific fragments thereof, or complements thereto.
 21. Anantibody which is specific-for an amino acid sequence selected from SEQID NO 2, 4, or 6.